Mutant fragments of OspA and methods and uses relating thereto

ABSTRACT

The present invention relates to compositions and methods for the prevention and treatment of  Borrelia  infection. Particularly, the present invention relates to a polypeptide comprising a hybrid C-terminal fragment of an outer surface protein A (OspA), a nucleic acid coding the same, an antibody specifically binding the same, a pharmaceutical composition (particularly for use as a medicament or in a method of treating or preventing a  Borrelia  infection) comprising the polypeptide and/or the nucleic acid and/or the antibody, a method of treating or preventing a  Borrelia  infection and a method of immunizing a subject.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/944,835, filed Apr. 4, 2018, now U.S. Pat. No. 10,766,931, which is acontinuation of U.S. application Ser. No. 15/110,151, now U.S. Pat. No.9,975,927, filed Jul. 7, 2016, which is a national stage filing under 35U.S.C. § 371 of international applicationPCT/EP2015/050365, filed Jan.9, 2015, which was published under PCT Article 21(2) in English, thecontent of each of which are incorporated by reference herein in itsentirety.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for theprevention and treatment of Borrelia infection. Particularly, thepresent invention relates to a polypeptide comprising a hybridC-terminal fragment of an outer surface protein A (OspA), a nucleic acidcoding the same, an antibody specifically binding the same, apharmaceutical composition (particularly for use as a medicament or in amethod of treating or preventing a Borrelia infection) comprising thepolypeptide and/or the nucleic acid and/or the antibody, a method oftreating or preventing a Borrelia infection and a method of immunizing asubject.

BACKGROUND OF THE INVENTION

Lyme borreliosis, or Lyme disease, is the most commonly reportedtick-borne disease in Europe and North America. The disease is caused byinfection with the arthropod-borne gram-negative-like spirochete,Borrelia burgdorferi sensu lato (B. burgdorferi s.l.), and can involvemultiple organs or tissues, resulting in skin, cardiac, musculoskeletaland neurological disorders. In most countries, Lyme borreliosis is not anotifiable disease; therefore, exact data regarding annual incidentrates are not available. In the United States, the causative agent is B.burgdorferi sensu stricto (B. burgdorferi s.s.) and Lyme borreliosis islocalized to north-eastern, mid-Atlantic and upper north-central states.In 2010, a total of about 30,000 cases of Lyme borreliosis were reportedto the US to the Centers for Disease Control and Prevention (CDC). Anupdated report by the CDC in 2013, which takes into account diagnosticdata from other sources, estimates that the actual number of new casesper year in the United States is closer to 300,000(http://www.cdc.gov/media/releases/2013/p0819-lyme-disease.html). InEurope, B. afzelii and B. garinii are the main causative agents of Lymeborreliosis, as well as B. burgdorferi s.s. and B. bavariensis, whichcontribute to a lesser extent depending on the geographic location. Theprevalence of Lyme borreliosis varies considerably in different Europeancountries with an overall increased prevalence from west to east. Inmuch of Europe, the number of reported cases of Lyme borreliosis hasincreased since the early 1990s (e.g., the Czech Republic, Estonia,Lithuania; see Lyme borreliosis in Europe, WHO report of 2006), and thegeographic distribution of cases has also expanded. Borrelia belongs tothe family Spirochaetaceae, which is subdivided into the medicallyimportant genera Treponema, Leptospira and Borrelia. B. burgdorferi s.l.is a spiral-shaped, vigorously motile gram-negative bacterium, about10-20 μm long and 0.2-0.5 μm wide, that grows under microaerophilicconditions. The spirochetal cell wall consists of a cytoplasmic membranesurrounded by peptidoglycan and several flagella and then by aloosely-associated outer membrane.

Lyme borreliosis generally occurs in stages characterized by differentclinical manifestations, with remissions and exacerbations. Stage 1,early infection, consists of a localized infection of the skin, followedwithin days or weeks by stage 2, disseminated infection, and months toyears later by stage 3, persistent infection. However, the infection isvariable; some patients have only localized infections of the skin,while others display only later manifestations of the illness, such asarthritis. Different clinical syndromes of Lyme borreliosis are alsocaused by infection with diverse B. burgdorferi s.l. species. B.burgdorferi s.s. more often causes joint manifestations (arthritis) andheart problems, B. afzelii causes mainly dermal symptoms (erythemamigrans; EM and acrodermatitis chronica atrophicans; ACA), whereas B.garinii is implicated in most cases of neuroborreliosis.

Localized infection—The most common symptom of stage 1 of an infectionis erythema migrans, which occurs in 70-80% of infected people. Thisskin lesion is often followed by flu-like symptoms, such as myalgia,arthralgia, headache and fever. These non-specific symptoms occur in 50%of patients with erythema migrans.

Disseminated infection—During stage 2, the bacteria move into the bloodstream from the site of infection to distal tissues and organs.Neurological, cardiovascular and arthritic symptoms that occur in thisstage include meningitis, cranial neuropathy and intermittentinflammatory arthritis.

Persistent infection—Stage 3 of the infection is chronic and occurs frommonths to years after the tick bite. The most common symptom in NorthAmerica is rheumatoid arthritis, caused by an infection with B.burgdorferi s.s. Persistent infection of the central nervous system withB. garinii causes more severe neurological symptoms during stage 3, anda persistent infection of the skin with B. afzelii results inacrodermatitis chronica atrophicans.

In some risk groups, such as farmers, forestry workers, hikers, runnersor vacationers, seroprevalence and disease incidence rates haveincreased, as well as in children under 15 years of age and adultsbetween 39 and 59, without gender preference. This increased incidenceof Lyme borreliosis is linked to changes in forest habitats as well associal factors. Environmental changes, such as forest fragmentation,have led to a sharp reduction of rodent predators such as foxes andbirds of prey, which in turn has led to an increase in the mousepopulation, with a subsequent increase in the tick population. Morerecently, patchy reforestation has increased the number of deer and thusthe number of ticks. Suburban sprawl and the increasing use of woodlandareas for recreation such as camping and hiking has brought humans intogreater contact with the larger number of tick Borrelia vectors. All ofthese factors together have contributed to a wider distribution ofBorrelia and a higher incidence of Lyme borreliosis.

Antimicrobial agents are the principle method of treatment of Borreliainfection. The antibiotic used depends on the stage of the disease,symptoms, and the patient's allergies to medication. The length of theantibiotic course also depends on the stage of the disease and theseverity of symptoms. Early Lyme borreliosis is typically treated withoral tetracyclines, such as doxycycline, and semi-synthetic penicillins,such as amoxicillin or penicillin V. Arthritic and neurologicaldisorders are treated with high-dose intravenous penicillin G orceftriaxone. Up to 30% of Lyme borreliosis patients do not display theearly characteristic symptoms of infection with Borrelia, makingdiagnosis and treatment problematic. The antibiotic course can be long(up to several months) and sometimes ineffective and is thus debated inthe Borrelia field, especially during later-stage disease. Even in thecase of effective treatment of Borrelia, patients can be left withdebilitating fatigue, pain, or neurological symptoms for yearsafterwards, which is referred to as post-treatment Lyme diseasesyndrome. In general, the use of antibiotics can have undesirableconsequences, such as the development of resistance by the targetmicro-organisms. Finally, antibiotic therapy may effectively cure Lymeborreliosis, but provides no protection against subsequent infections.

A monovalent serotype 1-OspA-based vaccine (LYMErix™) was approved andmarketed in the USA for the prevention of Lyme disease caused byBorrelia burgdorferi s.s., but the vaccine is no longer available.Furthermore, heterogeneity in OspA sequences across different serotypesin Europe and elsewhere precludes efficient protection with a vaccinebased on OspA from only a single serotype.

Chimeric OspA molecules comprising the proximal portion from one OspAserotype, together with the distal portion form another OspA serotype,while retaining antigenic properties of both of the parent polypeptides,may be used in the prevention and treatment of Lyme disease orborreliosis (WO2011/143617, WO2011/143623).

Currently, there is no preventative medicament for Lyme borreliosis onthe market and thus there is a need in the art for the development ofsuch a medicament that can provide effective protection against Borreliathat are present in the USA, Europe and elsewhere, especially for thedevelopment of a medicament that can provide effective protectionagainst several Borrelia serotypes simultaneously. The serotype 3OspA-containing heterodimer, Lip-S4D1-S3hybD1, of the current inventionimproves on our previously-disclosed heterodimer, Lip-S4D1-S3D1 (seeWO2014/006226) with regard to both ease of production and stimulation ofspecific antibodies as measured by antibody surface binding to serotype3 Borrelia spirochetes. Additionally, the more specific quality of theimmune response to the new heterodimer indicates that the potency isalso superior in comparison to the previously-disclosed heterodimer.

SUMMARY OF THE INVENTION

The present invention relates to a polypeptide comprising a hybridfragment of Borrelia outer surface protein A (OspA), a nucleic acidencoding the same, a vector which comprises such nucleic acid molecule,and a host cell comprising such vector. Furthermore, the inventionprovides a process for producing such polypeptide and a process forproducing a cell which expresses such polypeptide. Moreover, the presentinvention provides antibodies specifically binding to such polypeptide,a hybridoma cell producing such antibodies, methods for producing suchantibodies, a pharmaceutical composition comprising such polypeptide,nucleic acid molecule, vector or antibody, the use of such polypeptide,nucleic acid molecule, vector or antibody for the preparation of amedicament or a pharmaceutical composition (particularly for use as avaccine or in a method of treating or preventing a Borrelia infection),methods for diagnosing an infection and methods for treating orpreventing a Borrelia infection and methods of immunizing a subject. Inparticular, the invention relates to an improved polypeptide forimmunization against serotype 3 Borrelia, in that the polypeptide ismore favourable for purification and induces a more specific immuneresponse to Borrelia as assessed by surface binding to Borrelia whencompared with the serotype 3 OspA-containing construct disclosed in ourprevious application WO2014/006226.

Efforts to develop a subunit vaccine for prevention of Lyme borreliosishave been focused in large part on the use of borrelial outer surfaceprotein A (OspA) as an antigen. The OspA protein is expressed byBorrelia only when it is in the gut of the tick vector. Thus, OspAantibodies produced by vaccination do not fight infection in the body,but rather enter the gut of the tick when it takes a blood meal. There,the antibodies neutralise the spirochetes and block the migration ofbacteria from the midgut to the salivary glands of the tick, the routethrough which Borrelia enters the vertebrate host. Thus, OspA-specificantibodies prevent the transmission of Borrelia from the tick vector tothe human host.

The lipidated form of OspA from B. burgdorferi s.s., strain ZS7,together with aluminium hydroxide was commercially developed as avaccine against Borrelia (LYMErix™) by SmithKline Beecham, nowGlaxoSmithKline (GSK) for the US market. Three doses of LYMErix™ over aperiod of one year were needed for optimal protection. After the firsttwo doses, vaccine efficacy against Lyme borreliosis was 49%, and afterthe third dose, 76%. However, shortly after LYMErix™ was commerciallyavailable, it was withdrawn from the market in 2002. Reasons cited werematters of practical application of the vaccine, for example the needfor booster injections every year or every other year, as well as therelatively high cost of this preventive approach compared withantibiotic treatment of early infection. In addition, there was aconcern that LYMErix′ could trigger autoimmune reactions in a subgroupof the population due to sequence homology with a human protein,although this was never proven. In addition, cross-protection againstother clinically important Borrelia species was not provided by thisvaccine.

Accordingly, in one embodiment, it was an object of the presentinvention to provide an improved vaccine, e.g. for the prevention ofLyme borreliosis. Preferably, the vaccine is easily produced while beingprotective, safe and more effective than existing therapies and/orprovides protection against more than one Borrelia species.Additionally, it is well-known in the art that different patientsrespond differently to different vaccine compositions. Therefore, in anycase, it would be a distinct advantage to have alternative vaccinesavailable as additional options for vaccinations against Borrelia.

The problem underlying the present invention is solved by a polypeptidecomprising a hybrid C-terminal OspA fragment, wherein the hybridfragment consists of a C-terminal domain of an OspA protein of Borreliathat is comprised of a fragment derived from an OspA protein of aBorrelia strain different than B. garinii, strain PBr, and a secondfragment of OspA from B. garinii, strain PBr, and differs from thecorresponding wild-type sequence at least by the introduction of atleast one disulfide bond.

Surprisingly, the introduction of said hybrid C-terminal OspA fragmentcomprising a sequence from B. valaisiana, strain VS116, fused with asequence from serotype 3 OspA (B. garinii, strain PBr), with anintroduced disulfide bond, resulted in a heterodimer protein(Lip-S4D1-S3hybD1) that was easier to purify and stimulated a morespecific immune response to serotype 3 OspA than the heterodimerLip-S4D1-S3D1 of our previous invention (WO2014/006226). As shown in theExamples, the serotype 3 hybrid OspA C-terminal fragment of the presentinvention has a predicted electrostatic potential isocontour that ismore similar to other OspA fragments from other serotypes than theserotype 3 OspA fragment. Furthermore, the serotype 3 hybrid OspAC-terminal fragment-containing heterodimer (Lip-S4D1-S3hybD1) was easierto purify, requiring less steps to get a much higher yield than theLip-S4D1-S3D1 heterodimer of the previous invention. Additionally,although the observed antibody titers were similar, the antibodiesstimulated by the improved heterodimer combination vaccine bound morespecifically to Borrelia expressing serotype 3 OspA compared with theheterodimer combination vaccine of the previous invention. Finally, thein vivo protective capacity of the improved heterodimer combinationvaccine was high against the four Borrelia serotypes tested.

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, in a first aspect, the present invention relates to apolypeptide comprising a hybrid C-terminal fragment of an outer surfaceprotein A (OspA), wherein said hybrid C-terminal OspA fragment consistsof a C-terminal domain fusion of two different OspA of Borrelia strainsand differs from the corresponding wild-type fragment at least by theintroduction of at least one disulfide bond, e.g. a cystine. Inparticular, the hybrid C-terminal OspA fragment consists of a fusion ofamino acids from the OspA proteins from a strain different to B.garinii, strain PBr, e.g., B. valaisiana, strain VS116, or B.spielmanii; with amino acids from the OspA protein of B. garinii, strainPBr, with the introduction of at least one disulfide bond (cystine).Specifically, the polypeptide comprises a hybrid C-terminal OspA (outersurface protein A of Borrelia) fragment, wherein the hybrid C-terminalOspA fragment consists, from the N- to C-terminal direction, of i) afirst OspA portion consisting of amino acids 125-176 or amino acids126-175 of OspA from a Borrelia strain that is not the correspondingfragment of B. garinii, strain PBr, with SEQ ID NO: 8, and ii) a secondOspA portion consisting of amino acids 177-274 or amino acids 176-274,(but most preferably amino acids 177-274), of OspA from B. garinii,strain PBr (SEQ ID NO: 8), wherein the second OspA portion is mutant andcystine-stabilized in that it differs from the corresponding wild-typesequence at least by the substitution of the wild-type amino acid atposition 182 of SEQ ID NO: 8 by a cysteine and by the substitution ofthe wild-type amino acid at position 269 of SEQ ID NO: 8 by a cysteineand wherein a disulfide bond between the cysteine at position 182 andthe cysteine at position 269 of said second OspA fragment is present;and wherein the numbering of the amino acids and of the cysteinesubstitutions is according to the numbering of corresponding amino acidsof the full length OspA of B. burgdorferi s.s., strain B31 (SEQ ID NO:5).

Alternatively, the polypeptide comprises a hybrid C-terminal OspAfragment, wherein the hybrid C-terminal OspA fragment consists, from theN- to C-terminal direction, of

-   -   i) a first OspA fragment (also referred to as a first OspA        portion) consisting of amino acids 125-176 or amino acids        126-175 of OspA from a Borrelia strain that is not the        corresponding fragment of B. garinii, strain PBr, with SEQ ID        NO: 8, and    -   ii) a second OspA fragment (also referred to as a second OspA        portion) consisting of amino acids 177-274 of OspA from B.        garinii, strain PBr (SEQ ID NO: 8), wherein the second OspA        fragment differs from the corresponding wild-type sequence by        the substitution of the wild-type amino acid at position 182 of        SEQ ID NO: 8 by a cysteine and by the substitution of the        wild-type amino acid at position 269 of SEQ ID NO: 8 and wherein        a disulfide bond between the cysteine at position 182 and the        cysteine at position 269 of said second OspA fragment is        present; and wherein the numbering of the cysteine substitutions        is according to the numbering of corresponding amino acids of        the full length OspA of B. burgdorferi s.s., strain B31 (SEQ ID        NO: 5).

It has been found that a polypeptide according to the invention can beeasily and effectively produced (see Example 2). Its production resultedin an increased yield in comparison to known products (see Example 2)Immunization with a polypeptide according to the invention producedhigher levels of antibodies specific to the OspA protein in its nativeform and is thus an improved vaccine (see Example 3). Moreover, itprovided protection against in vivo Borrelia challenge (see Example 4).

Borrelia is a genus of bacteria of the spirochete phylum. It causesborreliosis, a zoonotic, vector-borne disease transmitted primarily byticks and some by lice, depending on the species. At present there are36 known species of Borrelia. Of the 36 known species of Borrelia, 13 ofthese species are known to cause Lyme disease or borreliosis and aretransmitted by ticks. The major Borrelia species causing Lyme diseaseare Borrelia burgdorferi, Borrelia afzelii, and Borrelia garinii. Theterm B. burgdorferi s.l. encompasses at least 13 Borrelia species (TableA-1). These species occur in different geographic regions, and live innature in enzootic cycles involving ticks of the Ixodes ricinus complex(also called Ixodes persulcatus complex) and a wide range of animalhosts. Four Borrelia species are responsible for the majority ofinfections in humans: B. burgdorferi s.s., B. afzelii, B. bavariensisand B. garinii. Three other species, B. lusitaniae, B. bissettii and B.spielmanii, have occasionally been detected in humans, but their role inLyme borreliosis is uncertain at present. New species of Borrelia arestill being identified.

TABLE A-1 Principal tick vector Location Pathogenic species (4) Borreliaburgdorferi Ixodes scapularis Northeastern/north-central US (Borreliaburgdorferi s.s.) Ixodes pacificus Western US Ixodes ricinus EuropeIxodes persulcatus Asia Borrelia garinii Ixodes ricinus Europe Ixodespersulcatus Asia Borrelia afzelii Ixodes ricinus Europe Ixodespersulcatus Asia Borrelia bavariensis Ixodes ricinus Europe Ixodespersulcatus Asia Minimally pathogenic or non- pathogenic species (9)Borrelia andersonii Ixodes dentatus Eastern US Borrelia bissettii Ixodesspinipalpis Western US Ixodes pacificus Europe Ixodes ricinus Borreliavalaisiana Ixodes ricinus Europe and Asia Ixodes columnae Borrelialusitaniae Ixodes ricinus Europe Borrelia spielmanii Ixodes ricinusEurope Borrelia japonica Ixodes ovatus Japan Borrelia tanukii Ixodestanuki Japan Borrelia turdi Ixodes turdus Japan Borrelia sinica Ixodespersulcatus China

As detailed above, Borrelia outer surface protein A (OspA) is anabundant immunogenic lipoprotein of Borrelia of particular interestbecause of its potential as a vaccine candidate. OspA of B. burgdorferis.l. is a basic lipoprotein that has a molecular mass of approximately30 kDa and is encoded on a linear plasmid. An important aspect of theOspA protein is its N-terminal lipidation; that is, fatty acids with achain length of between C14 and C19 with or without double bonds areattached to the N-terminal cysteine residue, a feature that enhances theimmunogenicity of the OspA protein. It has been shown thatpoorly-immunogenic synthetic peptides induce stronger antibody responseswhen lipidated; for example, when covalently coupled to Pam₃Cys (Besslerand Jung, Research Immunology (1992) 143:548-552), a fatty acidsubstitution found at the amino terminus of many bacterial lipoproteinsthat are synthesized with a signal sequence specifying lipid attachment.Additionally, the Pam₃Cys moiety was shown to enhance immune responsesto OspA in mice, partially through its interaction with TLR-2/1 (Yoder,et al. (2003) Infection and Immunity 71:3894-3900). Therefore,lipidation of a C-terminal fragment of OspA would be expected to enhancethe immunogenicity and protective capacity of the fragment.

Analysis of isolates of B. burgdorferi s.l. obtained in North Americaand Europe has revealed that OspA has antigenic variability and thatseveral distinct groups can be defined based on serology. Anti-OspA mAbswhich bind to specific N- and C-terminal antigenic determinants havebeen reported. X-ray crystallography and NMR analysis have been used toidentify immunologically important hypervariable domains in OspA andhave mapped the LA-2 epitope to C-terminal amino acids 203-257 (Ding etal., Mol. Biol. 302: 1153-64, 2000). Previous studies have shown thatthe production of antibodies against the C-terminal epitope LA-2correlates with protective immunity after vaccination with OspA (VanHoecke et al. Vaccine (1996) 14(17-18):1620-6 and Steere et al., N EnglJ Med (1998) 339:209-215). Antibodies to LA-2 were shown to block thetransmission of Borrelia from tick to host (Golde et al., Infect Immun(1997) 65(3):882-889). These studies suggested that the C-terminalportion of the OspA protein may be sufficient for inducing protectiveimmunity. It should be noted that the sequence of the C-terminal portionof OspA is less highly-conserved between Borrelia serotypes than is theN-terminal portion (see FIG. 1 ).

Based on information from the studies outlined above, along with others,truncated forms of OspA comprising the C-terminal portion (also referredto herein as “OspA fragment” or “monomer”) were used in the previousinvention (WO2014/006226). The truncated forms of OspA proved to be lessprotective than the full-length OspA protein. Surprisingly, however, itwas found in the course of the previous invention that the introductionof a disulfide bond in the truncated form (also referred to herein as“mutant OspA fragment”, “cystine-stabilized OspA fragment”, or “mutantfragment” or “cystine-stabilized fragment”) overcomes this disadvantage.While not being limited to a specific mechanism, it is thought thatimproved protection is due to increased stability of the OspA fragment,as shown in assays measuring thermal stability.

In accordance with the previous invention, the mutant OspA fragment (inthe present invention also referred to as second OspA fragment) may bederived from any Borrelia species; however, due to their prevalence inthe medical field, particularly for humans, B. burgdorferi s.s., B.afzelii, B. bavariensis and B. garinii are preferred. In accordance withthe present invention, the first OspA portion is from any Borreliastrain except B. garinii, strain PBr and the second portion is from B.garinii, strain PBr. Therefore, the hybrid OspA fragment is derived froma fusion of amino acids from the OspA of B. garinii, strain PBr withamino acids from the OspA from any Borrelia species except B. garinii,strain PBr (and therefore an amino acid sequence different from thatamino acids from the OspA from B. garinii, strain PBr), particularlyfrom B. burgdorferi s.s., B. afzelii, and B. bavariensis, especially B.valaisiana, strain VS116. The first OspA portion consists of amino acids125-176 or amino acids 126-175 of OspA from a Borrelia strain that isnot the corresponding fragment of B. garinii, strain PBr, with SEQ IDNO: 8, and the second OspA portion consists of amino acids 176-274 ormost preferably amino acids 177-274 of OspA from B. garinii, strain PBr(SEQ ID NO: 8), wherein the second OspA portion is mutant andcystine-stabilized in that it differs from the corresponding wild-typesequence at least by the substitution of the wild-type amino acid atposition 182 of SEQ ID NO: 8 by a cysteine and by the substitution ofthe wild-type amino acid at position 269 of SEQ ID NO: 8 by a cysteineand wherein a disulfide bond between the cysteine at position 182 andthe cysteine at position 269 of said second OspA fragment is present;and wherein the numbering of the amino acids and of the cysteinesubstitutions is according to the numbering of corresponding amino acidsof the full length OspA of B. burgdorferi s.s., strain B31 (SEQ ID NO:5). However, further mutations relative to the wild-type portions may bepresent in the first and second portions according to the invention (seealso below). In a preferred embodiment the above substitutions withcysteine at positions 182 and 269 are the only mutations relative to thewild-type. Alternatively, the cystine-stabilized amino acids 177-274from Borrelia garinii, strain PBr differs therefrom by the substitutionof the threonine residue at amino acid 233 of wild-type OspA of Borreliagarinii, strain PBr, with a proline residue only.

Preferred examples of polypeptides comprise

-   -   a hybrid C-terminal OspA fragment consisting of amino acids        125-176 from B. valaisiana, strain VS116, and the        cystine-stabilized amino acids 177-274 from Borrelia garinii,        strain PBr (SEQ ID NO: 1), or    -   a hybrid C-terminal OspA fragment consisting of amino acids        126-175 from B. spielmanii and the cystine-stabilized amino        acids 177-274 from Borrelia garinii, strain PBr (SEQ ID NO: 51).

Therefore, in a preferred embodiment of the invention, the polypeptidecomprises or consists of the amino acid sequence of SEQ ID NO: 1.Alternatively, the polypeptide comprises or consists of the amino acidsequence of SEQ ID NO: 51.

In one embodiment of the present invention, the second OspA portion isidentical to the cystine-stabilized amino acids 177-274 from Borreliagarinii, strain PBr, but differs therefrom by the substitution of thethreonine residue at amino acid 233 of wild-type OspA of Borreliagarinii, strain PBr, with a proline residue (SEQ ID NO: 7).

The four Borrelia species B. burdorferi s.s., B. afzelii, B. bavariensisand B. garinii can be further classified according to their OspAserotypes, which have been determined by analysis with monoclonalantibodies specific to the respective OspA protein. Serotypes 1-7, whichaccount for the majority of human Borrelia infections, along with theirrates of prevalence, are shown in Table A-2 below.

TABLE A-2 Serotype designation and prevalence of B. burdorferi s.s., B.afzelii, B. bavariensis and B. garinii. Borrelia isolated from humancerebrospinal fluid or skin or from tick vectors were serotyped byprobing whole-cell lysates with mouse monoclonal antibodies, eachspecific to a particular epitope of OspA (as described by Wilske et al.,J. of Clin Microbiol (1993) 31(2): 340-350 and presented by BaxterBioscience at “Climate change effect on ticks and tick-borne diseases”,Brussels, 6 Feb. 2009). OspA serotype Prevalence Strain defined by inhuman source for Seq Borrelia sp. mAb testing disease sequence ID No: B.burgdorferi s.s. 1 11% B31 5 B. afzelii 2 63% K78 6 B. garinii 3 1.5% PBr 7.8 B. bavariensis 4  4% PBi 9 B. garinii 5  6% PHei 10 B. garinii 613% DK29 11 B. garinii 7 0.5%  T25 12

The structure of the OspA protein from B. burgdorferi s.s. strain B31was determined by Li et al. (Proc Natl Acad Sci (1997) 94:3584-3589). Itis composed of N-terminal (α-strands 1 to 4) and central β-sheets(α-strands 5 to 14n [N-terminal part]), barrel sheet 1 (α-strands 14c[C-terminal part] to 16), barrel sheet 2 (α-strands 17 to 21) and aC-terminal α-helix. The term “C-terminal OspA fragment” or “OspAC-terminal domain” or “C-terminal domain” or “wild-type fragment” or“C-terminal portion” with respect to OspA as used throughout the presentspecification shall mean the C-terminal amino acid sequence of OspA,i.e., OspA lacking at least the N-terminal β-sheet (including α-strands1 to 4). In OspA from B. burgdorferi s.s. strain B31, the N-terminalsheet consists of amino acids 17 to 70 (following post-translationalcleavage of the 16 amino acid long lipidation signal peptide).

The C-terminal OspA fragment of the current invention may also include alipidation signal sequence at the N-terminus, e.g., the lipidationsignal sequence of amino acids 1 to 16 of OspA (SEQ ID NO: 13) or OspB(SEQ ID NO: 14) from B. burgdorferi s.s. strain B31, a lipidation signalsequence from E. coli, referred to herein as the “lpp lipidation signal”(SEQ ID NO: 15), or any other signal sequence, e.g., as defined below.

Lipidation of a protein with an N-terminal lipidation signal sequence,such as those present on a nascent OspA polypeptide, occurs in the E.coli expression vector by the step-wise action of the enzymesdiacylglyceryl transferase, signal peptidase II and transacylase,respectively. The first step is the transfer of a diacylglyceride to thecysteine sulfhydryl group of the unmodified prolipoprotein, followed bythe cleavage of the signal peptide by signal peptidase II and, finally,the acylation of the □-amino group of the N-terminal cysteine of theapolipoprotein. The result is the placement of one lipid and a glycerolgroup with two further lipids attached on the N-terminal cysteineresidue of the polypeptide. The lipidation signal sequence, which iscleaved off during lipidation, is not present in the final polypeptidesequence.

Polypeptides are biological molecules of chains of amino acid monomerslinked by peptide (amide) bonds. The covalent chemical bonds are formedwhen the carboxyl group of one amino acid reacts with the amino group ofanother. Polypeptides of the present invention are continuous andunbranched amino acid chains. The polypeptide of the present inventioncomprises a cystine bond. According to the current invention, the mutantOspA fragment may be a lipidated protein, also lipoprotein, wherein thelipid moieties, along with the glycerol group, is also referred to as“Lip”. According to the invention, Lip comprises one to three lipidssuch as C₁₄₋₂₀ alkyl and/or C₁₄₋₂₀ alkenyl attached to a glycerol and anamino group of the N-terminal cysteine of the polypeptide of theinvention, or preferably wherein Lip is a moiety of formula (I) below,

in which one of R₁, R₂ or R₃ is C₁₄-C₂₀ alkyl or alkenyl, and each ofthe others, independently, is C₁₄-C₂₀alkyl or C₁₄-C₂₀ alkenyl, and X isan amino acid sequence attached to the cysteine residue shown in Formula(I). More preferably, Lip plus the N-terminal cysteine of thepolypeptide is N-palmitoyl-S-(2RS)-2,3-bis-(palmitoyloxy) propylcysteine (referred to herein as “Pam₃Cys”) and is connected via thecarbonyl C of the cysteine to said amino acid sequence of the invention.In Formula (I) above, R₁, R₂ and R₃ would be palmitoyl moieties and X isan amino acid sequence attached to the cysteine residue.

In accordance with the current invention, the C-terminal domain of anOspA may lack at least the N-terminal domain homologous to amino acids17 to 70 of OspA from B. burgdorferi s.s., strain B31. Additionally, theOspA C-terminal domain according to the present invention may also lackfurther portions of the central sheet as defined by Li and co-workers(Li et al., supra), particularly further strands such as the amino acidportions from amino acid 17 to 82, 93, 105, 118 or 119, preferably 17 to129, more preferably 1 to 124, 1 to 125, 1 to 129 or 1 to 130 of anyBorrelia, particularly B. burgdorferi s.s., strain B31, or homologousportions of an OspA protein from a Borrelia sp. other than B.burgdorferi s.s., strain B31.

In the context of the present invention, the OspA C-terminal domain isalso referred to as “OspA fragment” or “fragment of OspA”.

The “mutant C-terminal OspA fragment” or “mutant fragment” or “mutantOspA fragment” in the context of the polypeptide of the presentinvention and as used throughout the present specification shall meanthe OspA C-terminal fragment, as defined above and herein, which differsfrom the wild-type fragment at least by at least two introducedcysteines that can form a disulfide bond; i.e., a cystine. Without beingbound to that theory, it is assumed that the disulfide bond stabilizesthe fragment in a conformation conducive to the induction of antibodybinding. The fold of the wild-type C-terminal fragment of OspA showsreduced temperature stability in comparison to the full-length protein(Koide et al., Structure-based Design of a Second-generation LymeDisease Vaccine Based on a C-terminal Fragment of Borrelia burgdorferiOspA, J. Mol. Biol. (2005) 350:290-299). For the present invention, thesequence of the C-terminal domain of the B. burgdorferi s.s., strain B31OspA has been in silico analyzed to determine positions for introduceddisulfide bridges that may enhance the stability of the fold of thisC-terminal domain. The results of the analysis have been transferred tohomologous OspA fragments of other Borrelia species with the assumptionthat the fold is conserved across species.

The “hybrid C-terminal OspA fragment” or “hybrid fragment” or “hybridOspA fragment” in the context of the polypeptide of the presentinvention and as used throughout the present specification shall meanthe OspA C-terminal fragment, as defined above and herein, which differsfrom the wild-type fragment in that

-   -   a) the hybrid C-terminal OspA fragment consists of a fusion of        amino acids from a first OspA protein from a strain different        to B. garinii, strain PBr, e.g. B. valaisiana, strain VS116,        or B. spielmanii (referred to as first OspA portion); with amino        acids from a second OspA protein of B. garinii, strain PBr, with        the introduction of at least one disulfide bond (cystine)        (referred to as second OspA portion) and optionally one or more        further mutations; and    -   b) the at least two introduced cysteines in the hybrid        C-terminal OspA fragment form a disulfide bond, i.e. a cystine,        and wherein said introduced cysteines are introduced as        described herein and in accordance with the teaching of        WO2014/006226; and        wherein the hybrid C-terminal OspA fragment results in a        naturally folded fragment as measured e.g. by the efficiency of        binding of antibodies resulting from the vaccination by this        hybrid C-terminal OspA fragment in e.g. mice to Borrelia        serotype 3 antigen, e.g. by testing the binding of said        antibodies (obtained after three immunizations) to the surface        of serotype 3 Borrelia by flow cytometry, e.g. as described in        the Examples.

Typically, the disulfide bond may be introduced by the introduction ofone or more, preferably two, cysteine residues, wherein a disulfide bond(S—S bridge) is formed between the thiol groups of two cysteine residuesforming the amino acid cystine. Only one cysteine residue need beintroduced if a disulfide bond is formed with a cysteine residue presentin the wild-type OspA fragment. The two cysteines are introduced byamino acid substitution. Substitutions are a) at position 182 of theamino acid of the relevant wild-type OspA amino acid sequence by acysteine and b) at position 269 of the amino acid of the relevantwild-type OspA amino acid sequence by a cysteine and wherein a disulfidebond between the cysteine at position 182 and the cysteine at position269 of said OspA fragment is present forming thus a cystine. Thenumbering of the cysteine substitutions is according to the numbering ofcorresponding amino acids of the full length OspA of B. burgdorferis.s., strain B31 (SEQ ID NO: 5).

The mutant or hybrid OspA fragment may also comprise further mutationsrelative to the wild-type. As detailed above, the structure and surfacedomain of OspA are known in the art. Accordingly, the mutant fragmentmay comprise further mutations, particularly at sites not on the surfaceof the protein and/or not involved in the immune response and, thereforenot impacting antigenic capacity. These can include one or more aminoacid deletion(s), particularly small (e.g., up to 10, 9, 8, 7, 6, 5, 4,3, 2 or 1 amino acids) deletions, one or more amino acid addition(s)(particularly C- or N-terminally), one or more amino acidsubstitution(s), particularly one or more conservative amino acidsubstitutions. Preferably, the number of further mutations in the firstand second portion relative to the respective wild-type is at most 10,9, 8, 7, 6, 5, 4, more preferably 3 or 2, especially 1. More preferablythe further mutation(s) is/are in the second OspA portion only.Preferred mutations are substitutions, especially conservativesubstitutions. Examples of conservative amino acid substitutionsinclude, but are not limited to, those listed below:

Ala Ser Arg Lys Asn Gln; His Asp Glu Cys Ser Gln Asn Glu Asp His Asn;Gln Ile Leu, Val Leu Ile; Val Lys Arg; Gln; Asn Met Leu; Ile Phe Met;Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu

Preferred mutations include changes in selected portions of thefragment, for example, wherein the sequence with sequence similarity tohuman leukocyte function-associated antigen (hLFA-1), which exists in B.burgdorferi s.s. is modified, for example, replaced by a homologoussequence from an OspA protein from another Borrelia sp. The rationalefor this modification is to reduce the risk for inducing immunologicalcross-reaction with human proteins. Another preferred mutation is thesubstitution of a proline for the threonine at position 233 in the OspApolypeptide sequence from B. garinii, strain PBr (SEQ ID NO: 8). Alsopossible is the addition of a signal sequence for lipidation in thefinal, or an intermediate, fragment, or the addition of a marker protein(e.g., for identification or purification).

In some embodiments, the mutant or hybrid OspA fragment has an aminoacid sequence that has 60%, preferably at least 70%, more preferably atleast 80%, more preferably at least 85%, more preferably at least 90%,even more preferably at least 95% sequence identity to the wild-typefragment. In another embodiment, the sequence differs by at most 10%, atmost 9%, at most 8%, at most 7%, at most 6%, 5%, 4%, 3%, 2%, mostpreferably at most 1%, due to a sequence addition, deletion orsubstitution.

Identity, as known in the art and as used herein, is the relationshipbetween two or more polypeptide sequences, as determined by comparingthe sequences. In the art, identity also means the degree of sequencerelatedness between polypeptide or polynucleotide sequences, as the casemay be, as determined by the match between strings of such sequences.Identity can be readily calculated. While a number of methods exist tomeasure identity between two polynucleotides or two polypeptidesequences, the term is well known to skilled artisans (e.g. SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press, 1987).Preferred methods to determine identity are designed to give the largestmatch between the sequences tested. Methods to determine identity arecodified in computer programs Preferred computer program methods todetermine identity between two sequences include, but are not limitedto, the GCG program package (Devereux, J. et al., 1984), BLASTP, BLASTN,and FASTA (Altschul, S. et al., 1990).

In contrast to the mutant or hybrid OspA fragment, the “wild-typefragment” or “wild-type OspA fragment” in the context of the presentinvention relates to a fragment of a naturally-occurring OspA ofBorrelia. The wild-type fragment is obtained by N-terminal deletions,but it does not comprise internal deletions (except from signalsequences as detailed herein) or mutations. In relation to the hybridand mutant OspA fragment, the wild-type fragment consists of anidentical part of the OspA (identical length and same strain of OspA,etc.) and differs only in the alteration(s) detailed above.

The Polypeptides of the Invention

A polypeptide is a single linear polymer of amino acids linked bypeptide bonds, in some cases also by disulfide bonds. In accordance withthe present invention, the polypeptide may also comprise one or moreposttranslational modifications; i.e., an attached biochemicalfunctional group, such as an attached acetate, phosphate, lipid orcarbohydrate, preferably a lipid or lipids attached to the N-terminalcysteine along with a glycerol, more preferably 1 to 3 C₁₄-C₂₀ alkyl oralkenyl moieties, even more preferably 1 to 3 palmitoyl groups, mostpreferably three palmitoyl groups (Pam₃).

The polypeptide of the present invention is as defined above andcomprises or consists of a hybrid C-terminal OspA fragment, wherein thehybrid C-terminal OspA fragment consists, from the N- to C-terminaldirection, of

-   -   i) a first OspA portion consisting of amino acids of a first        C-terminal part of OspA from a Borrelia strain that is not the        corresponding fragment of B. garinii, strain PBr, with SEQ ID        NO: 8, and starts at a position around 125 or 126 and ends at        position 175 or 176; and    -   ii) a second OspA portion consisting of a second C-terminal part        of OspA that continuously follows the first C-terminal part        (e.g. if the first C-terminal part ends at position 175, the        second C-terminal part continues at position 176; or if the        first C-terminal part ends at positions 176, the second        C-terminal part continues at positions 177 and so forth, however        it may also be that one or two or more up to 10 amino acids may        be deleted in the fusion area of the 2 C-terminal parts, e.g.        and most preferably if the first C-terminal part ends at        position 175, the second C-terminal part continues at position        177), wherein the second OspA fragment differs from the        corresponding wild-type sequence by the substitution of the        wild-type amino acid at position 182 of SEQ ID NO: 8 by a        cysteine and by the substitution of the wild-type amino acid at        position 269 of SEQ ID NO: 8 by a cysteine and wherein a        disulfide bond between the cysteine at position 182 and the        cysteine at position 269 of said second OspA fragment is present        forming a cysteine (also referred to as “cystine-stabilized OspA        fragment”); and        wherein the numbering of the amino acids and of the cysteine        substitutions is according to the numbering of corresponding        amino acids of the full length OspA of B. burgdorferi s.s.,        strain B31 (SEQ ID NO: 5).

In a further embodiment of the present invention, the polypeptidecomprises or consists of the hybrid C-terminal OspA fragment consistingof amino acids 125-176 from B. valaisiana, strain VS116, and thecystine-stabilized amino acids 177-274 from Borrelia garinii, strain PBr(SEQ ID NO: 1).

In a further embodiment of the present invention, the polypeptidecomprises or consists of the hybrid C-terminal OspA fragment consistingof amino acids 126-175 from B. spielmanii and the cystine-stabilizedamino acids 177-274 from Borrelia garinii, strain PBr (SEQ ID NO: 51).

In a further embodiment of the present invention, the polypeptide is asdefined herein, and wherein the second OspA portion is identical to thecystine-stabilized amino acids 177-274 from Borrelia garinii, strainPBr, but differs therefrom only by the substitution of the threonineresidue at amino acid 233 of wild-type OspA of Borrelia garinii, strainPBr, with a proline residue.

The polypeptides of the invention as defined above and further asdefined herein provide for antibody titers in immunized animals, whereinsaid antibodies show improved binding to their respective antigens insitu when compared to relevant prior art polypeptides (e.g. as definedin WO2014/006226). Preferably, a polypeptide comprising the hybridC-terminal OspA fragment of the invention effects at least a 1.5-foldincrease, preferably at least a 2-fold increase, more preferably atleast a 3-fold increase, even more preferably at least a 4-foldincrease, even more preferably at least a 5-fold increase, mostpreferably at least a 10-fold increase in fluorescence intensity,measured by flow cytometry and illicited by antibodies raised afterthree immunizations with the polypeptide in mice by binding to thesurface of serotype 3 Borrelia, in comparison to the fluorescenceintensity illicited by antibodies raised to a polypeptide comprising aC-terminal domain of an OspA protein of Borrelia which differs from thecorresponding wild-type OspA sequence by at least the addition of atleast one cysteine bond, more preferably the Lip-S4D1-S3D1 heterodimerprotein as defined by SEQ ID NO: 31, particularly wherein the increasemay be determined as described in Example 3.

Furthermore, in a further embodiment of the present invention, thepolypeptide of the invention as defined above and herein can be producedin higher yields with standard processes and require less purificationsteps when compared to relevant prior art polypeptides (e.g. as definedin WO2014/006226). Preferably, a polypeptide comprising the hybridC-terminal OspA fragment of the invention shows at least a 1.5-foldincrease, preferably at least a 2-fold increase, more preferably atleast a 3-fold increase, even more preferably at least a 4-foldincrease, even more preferably at least a 5-fold increase, mostpreferably at least a 10-fold increase in production yield, measured inmilligrams per gram biomass, in comparison to a polypeptide comprising aC-terminal domain of an OspA protein of Borrelia which differs from thecorresponding wild-type OspA sequence by at least the addition of atleast one cysteine bond, more preferably the Lip-S4D1-S3D1 heterodimerprotein as defined by SEQ ID NO: 31, particularly wherein the increasemay be determined as described in Example 2.

Preferably, a polypeptide comprising the hybrid C-terminal OspA fragmentof the invention requires less steps for purification than a polypeptidecomprising a C-terminal domain of an OspA protein of Borrelia whichdiffers from the corresponding wild-type OspA sequence by at least theaddition of at least one cysteine bond, more preferably theLip-S4D1-S3D1 heterodimer protein as defined by SEQ ID NO: 31; morespecifically, requires at least one less chromatography step.

In a further embodiment of the present invention, the polypeptidecomprises a) a hybrid C-terminal OspA fragment as defined above andherein, and b) a mutant OspA fragment.

In a further embodiment of the present invention, the polypeptide of theinvention comprises a) a hybrid C-terminal OspA fragment as definedherein, and b) a second OspA fragment, wherein said OspA fragment isC-terminal in that it consists of a C-terminal domain of an OspA proteinof Borrelia and is mutant and cystine-stabilized in that it differs fromthe corresponding wild-type OspA sequence at least by the substitutionof the amino acid at position 182 of the wild-type sequence by acysteine and by the substitution of the amino acid at position 269 ofthe wild-type sequence by a cysteine and wherein a disulfide bondbetween the cysteine at position 182 and the cysteine at position 269 ofsaid OspA fragment is present; and furthermore wherein said mutant OspAfragment starts at position 123, 124, or 125 and ends at position 273 or274; and furthermore wherein the numbering of the amino acids and of thecysteine substitutions is according to the numbering of correspondingamino acids of the full length OspA of B. burgdorferi s.s., strain B31(SEQ ID NO: 5). In one embodiment of the present invention, the secondOspA fragment may be any of the mutant C-terminal fragments of OspA asdefined above, e.g. in the context of the previous invention(WO2014/006226).

According to the present invention, said mutant OspA fragment and hybridfragment of the invention do not comprise (i) the N-terminal sheet asdefined above and (ii) optionally one or more further strands of thecentral sheet as defined above. However, the polypeptide may compriseone or more functional sequences such as a signal sequence, e.g., alipidation signal sequence or a posttranslational modification, such aslipidation.

In a further embodiment of the present invention, the polypeptide of thepresent invention consists of (i) one or more mutant OspA fragments,wherein at least one is a hybrid C-terminal OspA fragment according tothe present invention, optionally joined by linkers, e.g., as definedbelow or one or more mutant OspA fragments and a serotype 3 hybrid OspAC-terminal fragment and (ii) optionally one or more amino acidsheterologous to OspA, particularly a signal sequence and (iii)optionally a posttranslational modification, such as lipidation.

Thus, in a further embodiment of the present invention, the polypeptideof the invention as defined above and herein additionally may comprise:

-   -   i) a polypeptide that is lipidated or wherein the polypeptide        comprises a lipidation signal, preferably the E. coli-derived        lpp lipidation signal MKATKLVLGAVILGSTLLAG (SEQ ID NO: 15);        and/or    -   ii) a polypeptide that comprises a lipidation site peptide lead        by an N-terminal cysteine residue as a site for lipidation,        preferably CSS; and/or    -   iii) a polypeptide that comprises a linker between the hybrid        C-terminal OspA fragment and the second cysteine-stabilized OspA        fragment, particularly wherein said linker comprises e.g.        GTSDKNNGSGSKEKNKDGKYS (SEQ ID NO: 16).

In one embodiment of the present invention, the second C-terminal OspAfragment is a hybrid C-terminal fragment of OspA according to thepresent invention.

In a further embodiment of the present invention, the polypeptidecomprises or consists of the heterodimer of Lip-S4D1-S3hybD1 (SEQ ID NO:27). Therefore, in a further aspect, the present invention relates to apolypeptide comprising or consisting of an amino acid sequence of SEQ IDNO: 27 (heterodimer of Lip-S4D1-S3hybD1).

The polypeptide of the present invention has protective capacity. Asdetailed above, the introduction of a disulfide bond into the hybrid andhybrid/mutant OspA fragment but also the hybrid nature of the OspAfragment of the invention increases the protective capacity of thepolypeptide relative to a polypeptide comprising the respective fragmentwithout the disulfide bond(s) and hybrid nature of the OspA fragment. Insome embodiments, the protective capacity is increased by at least 10%,more preferably by at least 20%, more preferably by at least 30%, morepreferably by at least 40%, more preferably by at least 50%, morepreferably by at least 60%, more preferably by at least 70%, morepreferably by at least 80%, even more preferably by at least 90%relative to a polypeptide comprising the respective fragment without thedisulfide bond(s) and hybrid nature of the OspA fragment.

The term protective capacity describes the ability to protect a subjectagainst a Borrelia infection. With respect to the polypeptide of theinvention, protective capacity relates to the ability of the polypeptideto induce an immune response that protects a subject against a Borreliainfection. Protective capacity can be tested by administering to asubject the polypeptide in a manner to induce an immune reaction againstthe polypeptide. Thereafter, the subject may be challenged withBorrelia. The subject's reaction to the infection is monitored.Particularly, the presence of Borrelia in the subject may be determined.For example, the polypeptide is protective if Borrelia cannot bedetected in the subject. The presence of Borrelia can be determined bydetecting Borrelia-specific nucleic acids (e.g., by PCR) orBorrelia-specific antibodies (e.g., by ELISA or Western blot) or bydetecting Borrelia itself (e.g., culturing organs or tissues in growthmedium and verifying the presence of Borrelia by microscopy). Inparticular, the protective capacity (“pc”), reported as a percentage,for a particular dose is defined as follows:pc (%)=[(number of total tested subjects−number of Borrelia-infectedsubjects)/number of total tested subjects]×100

Differences in protective capacity (Δpc) may be determined by, e.g.comparing the protective capacity (pc) of a mutant OspA fragment with adisulfide bond(s) (pc [with bond]) to the protective capacity of an OspAfragment without a disulfide bond(s) (pc [w/o bond]). In accordance withthe present invention, the polypeptides to be compared differ only inthe introduction of at least one disulfide bond. The change inprotective capacity (Δpc) by the introduction of the disulfide bond(s)is determined as follows:Δpc=(pc[sample]−pc[control])e.g. Δpc=(pc[with bond]−pc[w/o bond])

If Δpc is greater than zero (>0), assuming all other parameters (e.g.,dose and assay) are the same, then the protective capacity of the sample(e.g. the mutant OspA fragment with a disulfide bond(s)) is better thanthe protective capacity of the control (e.g. the OspA fragment without adisulfide bond(s)). Conversely, if Δpc is less than zero (<0) andassuming all other parameters (e.g., dose and assay) are the same, thenthe protective capacity of the sample (e.g. the mutant OspA fragmentwith a disulfide bond(s)) is less than the protective capacity of thecomparison (e.g., the OspA fragment without a disulfide bond(s)).

Preferably, the polypeptide of the present invention is assessed for itsprotective capacity by an in vivo challenge assay wherein mice immunizedwith the polypeptide of the invention or with a placebo control arechallenged with Borrelia introduced into the immunized subjects with ahypodermic needle (Needle Challenge Method) or by introduction by a tickvector (Tick Challenge Method).

The Needle Challenge Method is carried out for the desired Borreliastrain (e.g., B. burgdorferi, strain ZS7) by subcutaneously introducingBorrelia at a dose between 20 and 50 times the Infectious Dose (ID)₅₀ tomice that are immunized with said first polypeptide of the first aspector with an appropriate placebo (negative) control, such as buffer oradjuvant alone and comparing the rates of infection in the challengedmice. The ID₅₀ is defined as the dose at which 50% of the challengedmice are infected. The dose of Borrelia is measured in numbers ofbacteria. The challenge dose can vary widely and is strain-dependent;therefore, the virulence of the strain must first be assessed bychallenge experiments for determination of ID₅₀. Four weeks after needlechallenge, blood and tissues are collected for readout methods todetermine the infection status. These readout methods can be e.g. VlsEELISA on sera or qPCR on collected tissues for identification ofBorrelia, as described herein, or other methods.

The Tick Challenge Method is carried out by applying at least one ticknymph (e.g., I. ricinus) infected with Borrelia (e.g., B. afzelii,strain IS1), to a mouse that is immunized with said first polypeptide ofthe first aspect; and b) applying at least one infected tick nymph to asecond mouse that is immunized with said second polypeptide of the firstaspect; and c) comparing the rates of infection in the two mice,generally six weeks after challenge. Preferably, the assay or test isdone with a group of mice per polypeptide to be tested. A suitable testis also described and illustrated in the Examples. Assessment ofinfection status can be done using VlsE ELISA on sera or qPCR on DNAisolated from collected tissues, or using other suitable methods.

In a preferred embodiment of the present invention, the products of theinvention such as, e.g. the polypeptides of the invention comprising thehybrid OspA fragment and preferably the mutant OspA C-terminal fragmentadministered 3 times to a subject at a dose of 30 μg, preferably 10 μg,preferably 5.0 μg, preferably 1.0 μg, preferably 0.3 μg or lower have aprotective capacity of 50% or more, preferably 60% or more, morepreferably 70% or more, more preferably 80% or more, more preferably 90%or more, even more preferably 95% or more, most preferred 99% or more.In one embodiment, the protective capacity is assessed in an in vivochallenge method, preferably a Tick Challenge Method, more preferably aNeedle Challenge Method, e.g. as described in the Examples.

In a preferred embodiment, the difference in protective capacity (Δpc)between the polypeptides of the invention comprising the hybrid OspAC-terminal fragment and the placebo (negative) control is at least 50%,especially at least 60%, preferably at least 70%, more preferably atleast 80%, even more preferably at least 90%, even more preferably atleast 95%, most preferably at least 99%, when administered 3 times to asubject at a dose of 30 μg, preferably 10 μg, preferably 5.0 μg,preferably 1.0 μg, preferably 0.3 μg or lower.

In accordance with the present invention, the first part of the hybridOspA may be from any Borrelia strain other than B. garinii, strain PBr,with SEQ ID NO: 8, particularly from those specified herein such as B.burgdorferi s.s., B. garinii (not strain PBr), B. afzelii, B. andersoni,B. bissettii, B. valaisiana, B. lusitaniae, B. spielmanii, B. japonica,B. tanukii, B. turdi or B. sinica, B. bavariensis, preferably from B.burgdorferi s.s., B. afzelii, B. bavariensis or B. garinii, or a fusionof OspA protein fragments from two or more of these species. Preferably,the OspA is from B. valaisiana, particularly strain VS116 (SEQ ID NO: 4)but may be from B. afzelii, particularly strain K78, OspA serotype 2(SEQ ID NO: 6); B. burgdorferi s.s., particularly strain B31, OspAserotype 1 (SEQ ID NO: 5); B. garinii, particularly strain PBr, OspAserotype 3 (SEQ ID NO: 8); B. bavariensis, particularly strain PBi, OspAserotype 4 (SEQ ID NO: 9); B. garinii, particularly strain PHei, OspAserotype 5 (SEQ ID NO: 10); B. garinii, particularly strain DK29, OspAserotype 6 (SEQ ID NO: 11) or B. garinii, particularly strain T25, OspAserotype 7 (SEQ ID NO: 12). The amino acid sequences of these OspAproteins (full-length) are given below.

In accordance with the present invention, the disulfide bond may also beformed between cysteines that have been introduced at any position ofthe OspA fragment allowing or supporting appropriate folding of thefragment. The positions may be selected, as detailed above, based on theknown structure of the OspA. In a preferred embodiment, the polypeptideof the current invention contains at least one disulfide bond introducedby the insertion of a cysteine residue at one of residues 182+/−3 andone of residues 269+/−3 (disulfide bond type 1; “D1”) of a B. afzelii,particularly B. afzelii K78 serotype 2 OspA, or the homologous aminoacids of an OspA from a Borrelia other than B. afzelii, such as B.burgdorferi s.s., particularly strain B31, serotype 1; B. garinii,particularly strain PBr, serotype 3; B. bavariensis, particularly strainPBi, serotype 4; B. garinii, particularly strain PHei, serotype 5; B.garinii, particularly strain DK29, serotype 6; B. garinii, particularlystrain T25, serotype 7 or a fusion of amino acids 125-176 of OspA of B.valaisiana, strain VS116, or amino acids 126-175 of OspA of B.spielmanii and amino acids 177-274 of B. garinii, strain PBr (SEQ ID NO:8).

It is noted that:

Position 182+/−3 is an abbreviation for position 179, 180, 181, 182,183, 184 or 185, preferably 182. Position 269+/−3 is an abbreviation forposition 266, 267, 268, 269, 270, 271 or 272, preferably 269.

In a preferred embodiment, the additional mutant fragment is derivedfrom the amino acids from position 125, 126, 130 or 131 to position 273of the wild-type sequence of the OspA of B. afzelii strain K78, serotype2 (SEQ ID NO: 6) and differs only by the introduction of at least onedisulfide bond, particularly wherein the at least one disulfide bond isbetween positions 182 and 269 (disulfide bond type 1); or the homologousfragments and positions of an OspA from a Borrelia sp. other than B.afzelii, such as B. burgdorferi s.s., particularly strain B31, serotype1; B. garinii, particularly strain PBr, serotype 3; B. bavariensis,particularly strain PBi, serotype 4; B. garinii, particularly strainPHei, serotype 5; B. garinii, particularly strain DK29, serotype 6 or B.garinii, particularly strain T25, serotype 7.

In a further embodiment, the mutant fragment may be an amino acidsequence selected from the group consisting of SEQ ID NO: 43, SEQ ID NO:44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, mostpreferably SEQ ID NO: 46, and an amino acid sequence that has at least80%, more preferably at least 85%, more preferably at least 90%, evenmore preferably at least 95% sequence identity to at least one ofsequences with SEQ ID NOs: 19 to 25, wherein the cysteines are notreplaced. Further details on mutations and sequence identity are givenabove.

As detailed above, the polypeptide of the present invention may comprisesignal sequences. It has been shown that lipidation confers adjuvantproperties on OspA. Accordingly, lipidated forms of the polypeptide ofthe invention or polypeptides comprising a lipidation signal arepreferred. In a preferred embodiment, the polypeptide of the currentinvention comprises a lipidation signal, preferably a lipidation signalof a Borrelia outer surface protein, OspA or OspB (SEQ ID NOs: 13 and14, respectively) or more preferably an E. coli lpp lipidation signalsequence (SEQ ID NO: 15). The OspA fragment of the invention comprisinga lipidation signal is lipidated during processing and the lipidationsignal peptide is cleaved off; therefore, the signal peptide is nolonger present in the mature lipidated protein.

Lipidated proteins according to the current invention are labeled with“Lip” at the N-terminus to indicate the addition of 3 fatty acid groupsand a glycerol to the polypeptide. Suitable lipidation signals asdescribed above include MKKYLLGIGLILALIA (SEQ ID NO: 13),MRLLIGFALALALIG (SEQ ID NO: 14) and MKATKLVLGAVILGSTLLAG (SEQ ID NO:15). Because lipid moieties and a glycerol are attached to theN-terminal cysteine residue which is present in the full-lengthwild-type OspA protein, OspA C-terminal fragments for lipidation mayadditionally comprise a peptide comprising a cysteine residue followedby additional amino acids. For example, sequences such as CSS or CKQN(SEQ ID NO: 62) immediately C-terminal to the lipidation signal sequenceprovide an N-terminal cysteine residue for lipidation upon cleavage ofthe lipidation signal peptide. The lipidated cysteine-containingpeptides are present in the final lipidated polypeptide of theinvention.

It has been speculated that the OspA protein of B. burgdorferi s.s.comprises a sequence with the capacity to bind to a T cell receptor thatalso has the capacity to bind to human leukocyte function-associatedantigen (hLFA-1) (herein referred to also as “hLFA-1-like sequence”).The similarity of this OspA region to hLFA-1 may result in an immuneresponse with cross-reactivity upon administration of B. burgdorferis.s. OspA to a human subject and may induce autoimmune diseases,particularly autoimmune arthritis, in susceptible individuals.Accordingly, in a preferred embodiment, the polypeptide of the currentinvention does not comprise a sequence with binding capacity to the Tcell receptor that has a binding capacity to the human leukocytefunction-associated antigen (hLFA-1), and particularly does not comprisethe amino acid sequence GYVLEGTLTAE (SEQ ID NO: 17). To this end, thehLFA-1-like sequence, particularly the amino acid sequence GYVLEGTLTAE(SEQ ID NO: 17), may be replaced with a homologous sequence from an OspAprotein of another Borrelia sp., particularly with NFTLEGKVAND (SEQ IDNO: 18).

In a preferred embodiment, the polypeptide of the current inventioncomprising at least one disulfide bond essentially establishes the sameprotective capacity with said polypeptide against a Borrelia infectionrelative to at least one of the wild-type full-length OspA proteinsderived from at least one Borrelia strain, particularly B. afzelii K78,OspA serotype 2 (SEQ ID NO: 6); B. burgdorferi s.s., particularly strainB31, serotype 1 (SEQ ID NO: 5); B. garinii, particularly strain PBr,serotype 3 (SEQ ID NOs: 7 and 8); B. bavariensis, particularly strainPBi, serotype 4 (SEQ ID NO: 9); B. garinii, particularly strain PHei,serotype 5 (SEQ ID NO: 10); B. garinii, particularly strain DK29,serotype 6 (SEQ ID NO: 11) or B. garinii, particularly strain T25,serotype 7 (SEQ ID NO: 12).

Please note that further details on mutations and sequence identity aregiven above.

TABLE A-3 Nomenclature and SEQ ID NOs. of the lipidated mutant OspAfragment heterodimers described in the current invention. Lipidatedmutant OspA fragment heterodimer* SEQ ID NO: Lip-S1D1-S2D1 29Lip-S5D1-S6D1 33 Lip-S4D1-S3D1 31 Lip-S4D1-S3hybD1 27 *S = Serotype(1-6) (see Table A-2); S3hyb = fusion of amino acids 125-176 of B.valaisiana and amino acids 177-274 of B. garinii, strain PBr D1 =Disulfide Bond Type 1(cysteine residues inserted at position 183 +/− 3and 270 +/− 3); Lip = lipidation: the N-terminal addition of glyceroland fatty acid residues.

In another preferred embodiment, the polypeptide according to the firstaspect comprises at least two or three mutant fragments which areconnected via one or more linkers. A linker is a rather short amino acidsequence employed to connect two fragments. It should be designed inorder to avoid any negative impact on the fragments, their interactionin subjects to be treated or vaccinated or upon their protectivecapacity. Preferred are short linkers of at most 21 amino acids,particularly at most 15 amino acids, especially at most 12 or 8 aminoacids. More preferably, the one or more linkers is/are composed of smallamino acids in order to reduce or minimize interactions with thefragments, such as glycine, serine and alanine. A preferred linker isthe “LN1” peptide linker, a fusion of two separate loop regions of theN-terminal half of OspA from B. burgdorferi s.s., strain B31 (aa 65-74and aa 42-53, with an amino acid exchange at position 53 of D53S) whichhas the following sequence: GTSDKNNGSGSKEKNKDGKYS (SEQ ID NO: 16). Thelinker may comprise a lipitation site.

In another preferred embodiment, the polypeptide according to the firstaspect comprises a polypeptide with a total size of at most 500 aminoacids, comprising two or three different mutant fragments as defined inpreferred embodiments of the first aspect; or a polypeptide whichconsists of essentially two or three different mutant fragments, one ortwo linkers and, optionally, an N-terminal cysteine; and/or apolypeptide which consists of essentially two or three different mutantfragments, an N-terminal extension of the fragment consisting of at most24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12 or 11 amino acids,preferably at most 10, 9, 8, 7 or 6 amino acids, still more preferablyat most 5, 4, 3, 2 or 1 amino acid(s), wherein the N-terminal extensionis located directly N-terminally from the fragment in the respectiveBorrelia OspA and, optionally, an N-terminal cysteine. The N-terminalcysteine may optionally be followed by a short peptide linker from 1-10amino acids long, and preferably takes the form of an N-terminal CSSpeptide.

The Nucleic Acids of the Invention and Related Aspects

In a further aspect, the present invention relates to a nucleic acidcoding for a polypeptide as defined herein and above in the context ofthe present invention. The nucleic acid may be comprised in a vectorand/or cell.

The invention further provides a nucleic acid encoding a polypeptide ofthe invention. For the purposes of the invention the term “nucleicacid(s)” generally refers to any polyribonucleotide orpolydeoxyribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA including single and double-stranded regions/forms.

The term “nucleic acid encoding a polypeptide” as used hereinencompasses polynucleotides that include a sequence encoding a peptideor polypeptide of the invention. The term also encompassespolynucleotides that include a single continuous region or discontinuousregions encoding the peptide or polypeptide (for example,polynucleotides interrupted by integrated phage, an integrated insertionsequence, an integrated vector sequence, an integrated transposonsequence, or due to RNA editing or genomic DNA reorganization) togetherwith additional regions, that also may contain coding and/or non-codingsequences.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are many nucleotidesequences that encode a polypeptide as described herein. Some of thesepolynucleotides bear minimal similarity to the nucleotide sequence ofany native (i.e., naturally occurring) gene. Nonetheless,polynucleotides that vary due to differences in codon usage arespecifically contemplated by the present invention, for examplepolynucleotides that are optimized for human and/or primate and/or E.coli codon selection.

Sequences encoding a desired polypeptide may be synthesized, in whole orin part, using chemical methods well known in the art (see Caruthers, M.H. et al., Nucl. Acids Res. Symp. Ser. pp. 215-223 (1980), Horn et al.,Nucl. Acids Res. Symp. Ser. pp. 225-232 (1980)). Alternatively, theprotein itself may be produced using chemical methods to synthesize theamino acid sequence of a polypeptide, or a portion thereof. For example,peptide synthesis can be performed using various solid-phase techniques(Roberge et al., Science 269:202-204 (1995)) and automated synthesis maybe achieved, for example, using the ASI 431 A Peptide Synthesizer(Perkin Elmer, Palo Alto, Calif.).

Moreover, the polynucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterpolypeptide encoding sequences for a variety of reasons, including butnot limited to, alterations which modify the cloning, processing, and/orexpression of the gene product. For example, DNA shuffling by randomfragmentation and PCR reassembly of gene fragments and syntheticoligonucleotides may be used to engineer the nucleotide sequences. Inaddition, site-directed mutagenesis may be used to insert newrestriction sites, alter glycosylation patterns, change codonpreference, produce splice variants, or introduce mutations, and soforth.

In a further aspect of the invention the present invention relates tovectors comprising a nucleic acid of the invention, e.g. linked to aninducible promoter such that when the promoter is induced a polypeptideencoded by the nucleic acid is expressed. In a preferred embodiment, thevector is pET28b(+) (http://www.addgene.org/vector-database/2566/).

A further aspect of the invention comprises said vector wherein theinducible promoter is activated by addition of a sufficient quantity ofIPTG (Isopropyl β-D-1-thiogalactopyranoside) preferably to the growthmedium. Optionally this is at a concentration of between 0.1 and 10 mM,0.1 and 5 mM, 0.1 and 2.5 mM, 0.2 and 10 mM, 0.2 and 5 mM, 0.2 and 2.5mM, 0.4 and 10 mM, 1 and 10 mM, 1 and 5 mM, 2.5 and 10 mM, 2.5 and 5 mM,5 and 10 mM. Alternatively the promoter may be induced by a change intemperature or pH.

Nucleic acid molecule as used herein generally refers to any ribonucleicacid molecule or deoxyribonucleic acid molecule, which may be unmodifiedRNA or DNA or modified RNA or DNA. Thus, for instance, nucleic acidmolecule as used herein refers to at least single- and double-strandedDNA, hybrid molecules comprising DNA and RNA that may be single-strandedor, more typically, double-stranded, or a mixture of single- anddouble-stranded regions. As used herein, the term nucleic acid moleculeincludes DNA or RNA molecules as described above that contain one ormore modified bases. Thus, DNA or RNA molecules with backbones modifiedfor stability or for other reasons are “nucleic acid molecule” as thatterm is intended herein. Moreover, DNA or RNA species comprising unusualbases, such as inosine, or modified bases, such as tritylated bases, toname just two examples, are also nucleic acid molecules as definedherein. It will be appreciated that a great variety of modificationshave been made to DNA and RNA molecules that serve many useful purposesknown to those of skill in the art. The term nucleic acid molecule asused herein embraces such chemically, enzymatically or metabolicallymodified forms of nucleic acid molecules, as well as the chemical formsof DNA and RNA characteristic of viruses and cells, including simple andcomplex cells, inter alia. The term nucleic acid molecule alsoencompasses short nucleic acid molecules often referred to asoligonucleotide(s). The terms “polynucleotide” and “nucleic acid” or“nucleic acid molecule” are used interchangeably herein.

The nucleic acids according to the present invention may be chemicallysynthesized. Alternatively, the nucleic acids can be isolated fromBorrelia and modified by methods known to one skilled in the art. Thesame applies to the polypeptides according to the present invention.

Furthermore, the nucleic acid of the present invention can befunctionally linked, using standard techniques such as cloning, to anydesired sequence(s), whether a Borrelia regulatory sequence or aheterologous regulatory sequence, heterologous leader sequence,heterologous marker sequence or a heterologous coding sequence to createa fusion gene.

Nucleic acid molecules of the present invention may be in the form ofRNA, such as mRNA or cRNA, or in the form of DNA, including, forinstance, cDNA and genomic DNA obtained by cloning or produced bychemical synthesis techniques or by a combination thereof. The DNA maybe triple-stranded, double-stranded or single-stranded. Single-strandedDNA may be the coding strand, also known as the sense strand, or it maybe the non-coding strand, also referred to as the anti-sense strand.

The nucleic acid of the present invention may be comprised in a vectoror in a host cell. Accordingly, the present invention also relates to avector or a host cell, preferably E. coli, comprising a nucleic acidmolecule according to the present invention. The vector may comprise theabove-mentioned nucleic acid in such a manner that the vector isreplicable and can express the protein encoded by the nucleotidesequence in a host cell.

For recombinant production of the polypeptides of the invention, hostcells can be genetically engineered to incorporate expression systems orportions thereof of the nucleic acid of the invention. Introduction of anucleic acid into a host cell can be effected by methods described inmany standard laboratory manuals, such as Davis, et al., BASIC METHODSIN MOLECULAR BIOLOGY, (1986) and Sambrook, et al., MOLECULAR CLONING: ALABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989), such as, calcium phosphate transfection,DEAE-dextran mediated transfection, transvection, microinjection,cationic lipid-mediated transfection, electroporation, conjugation,transduction, scrape loading, ballistic introduction and infection.

Representative examples of appropriate hosts include gram negativebacterial cells, such as cells of E. coli, Acinetobacter,Actinobacillus, Bordetella, Brucella, Campylobacter, Cyanobacteria,Enterobacter, Erwinia, Franciscella, Helicobacter, Hemophilus,Klebsiella, Legionella, Moraxella, Neisseria, Pasteurella, Proteus,Pseudomonas, Salmonella, Serratia, Shigella, Treponema, Vibrio,Yersinia. In one embodiment, the host cell is an Escherichia coli cell.In a preferred embodiment, the host cell is an E. coli BL21(DE3) cell oran E. coli BL21 Star™ (DE3) cell.

Alternatively, gram positive bacterial cells may also be used. A greatvariety of expression systems can be used to produce the polypeptides ofthe invention. In one embodiment the vector is derived from bacterialplasmids. Generally any system or vector suitable to maintain, propagateor express polynucleotides and/or to express a polypeptide in a host maybe used for expression in this regard. The appropriate DNA sequence maybe inserted into the expression system by any of a variety of well-knownand routine techniques, such as, for example, those set forth inSambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL (supra).

In one embodiment of the current invention, the cells are grown underselective pressure, such as in the presence of antibiotics, preferablykanamycin. In another embodiment, cells are grown in the absence ofantibiotics.

A great variety of expression vectors can be used to express thepolypeptides according to the present invention. Generally, any vectorsuitable to maintain, propagate or express nucleic acids to express apolypeptide in a host may be used for expression in this regard. Inaccordance with this aspect of the invention the vector may be, forexample, a plasmid vector, a single- or double-stranded phage vector ora single- or double-stranded RNA or DNA viral vector. Starting plasmidsdisclosed herein are either commercially available, publicly available,or can be constructed from available plasmids by routine application ofwell-known, published procedures. Preferred among vectors, in certainrespects, are those for expression of nucleic acid molecules and thepolypeptides according to the present invention. Nucleic acid constructsin host cells can be used in a conventional manner to produce the geneproduct encoded by the recombinant sequence. Alternatively, thepolypeptides according to the present invention can be syntheticallyproduced by conventional peptide synthesizers.

In addition, the present invention relates to a host cell comprisingthis vector. Representative examples of appropriate host cells includebacteria, such as streptococci, staphylococci, E. coli, Streptomyces andBacillus subtilis; fungi, such as yeast and Aspergillus; insect cellssuch as Drosophila S2 and Spodoptera Sf9 cells; mammalian cells such asCHO, COS, HeLa, C127, 3T3, BHK, 293 or Bowes melanoma cells; and plantcells. Cell-free translation systems can also be employed to producesuch proteins using RNA derived from the DNA construct of the presentinvention.

In order to express the desired amino acid sequence practically byintroducing the vector according to the present invention into a hostcell, the vector may contain, in addition to the nucleic acid sequenceaccording to the present invention, other sequences for controlling theexpression (e.g., promoter sequences, terminator sequences and enhancersequences) and gene markers for selecting microorganisms, insect cells,animal culture cells, or the like (e.g., neomycin resistance genes andkanamycin resistance genes). Furthermore, the vector may contain thenucleic acid sequence according to the present invention in a repeatedform (e.g., in tandem). The vector may be constructed based onprocedures which are conventionally used in the field of geneticengineering.

The host cells may be cultured in an appropriate medium, and the proteinaccording to the present invention may be obtained from the cultureproduct. The protein according to the present invention may be recoveredfrom the culture medium and purified in the conventional manner.

Accordingly, the present invention also relates to a process forproducing a cell which expresses a polypeptide according to the presentinvention, comprising transforming or transfecting a suitable host cellwith the vector according to the present invention or a process forproducing the polypeptide according to the present invention, comprisingexpressing the nucleic acid molecule according to the present invention.

Alternatively, a method for producing a polypeptide as defined above maybe characterized by the following steps:

-   -   a) introducing a vector encoding the polypeptide into a host        cell,    -   b) growing the host cell under conditions allowing for        expression of said polypeptide,    -   c) homogenizing said host cell, and    -   d) subjecting the host cell homogenate to purification steps.

The invention further relates to a method for producing a polypeptide asdefined above, characterized by the following steps:

-   -   a) introducing a nucleic acid encoding a polypeptide into a        vector,    -   b) introducing said vector into a host cell,    -   c) growing said host cell under conditions allowing for        expression of polypeptide,    -   d) homogenizing said host cell,    -   e) enriching polypeptide in the lipid phase by phase separation,        and    -   f) further purifying over a gel filtration column.

The invention further relates to a method for producing a polypeptide asdefined above, characterized by the following steps:

-   -   a) introducing a nucleic acid encoding a polypeptide into a        vector,    -   b) introducing said vector into a host cell,    -   c) growing said host cell under conditions allowing for        expression of polypeptide,    -   d) homogenizing said host cell,    -   e) enriching polypeptide in the lipid phase by phase separation,    -   g) purifying over a gel filtration column, and    -   h) optionally, further processing over a buffer exchange column.        The Antibodies of the Invention and Related Aspects

The problem underlying the present invention is solved in a furtheraspect by an antibody, or at least an effective part thereof, whichselectively binds to the hybrid C-terminal OspA fragment as defined inthe context of the present invention, but not to the first OspA portionand the second OspA portion alone (i.e. if not fused to the otherportion).

In a preferred embodiment the antibody is a monoclonal antibody.

In another preferred embodiment said effective part comprises an Fabfragment, an F(ab) fragment, an F(ab)N fragment, an F(ab)₂ fragment oran F_(v) fragment or single domain antibody.

In still another embodiment of the invention the antibody is a chimericantibody.

In yet another embodiment the antibody is a humanized antibody.

In a preferred aspect, antibodies of the invention bind specifically tohybrid OspA fragment polypeptides of the invention, but not to the firstOspA portion and the second OspA portion alone and not to correspondingwild-type OspA fragment polypeptides. In a more preferred aspect, theantibody binds specifically to the disulfide bond of the mutant OspAfragment of the invention.

The term “specificity” refers to the number of different types ofantigens or antigenic determinants to which a particular antigen-bindingmolecule or antigen-binding protein (such as a Nanobody or a polypeptideof the invention) can bind. The specificity of an antigen-bindingprotein can be determined based on affinity and/or avidity. Theaffinity, represented by the equilibrium constant for the dissociationof an antigen with an antigen-binding protein (K_(D)), is a measure forthe binding strength between an antigenic determinant and anantigen-binding site on the antigen-binding protein: the lesser thevalue of the K_(D), the stronger the binding strength between anantigenic determinant and the antigen-binding molecule (alternatively,the affinity can also be expressed as the affinity constant (K_(A)),which is 1/K_(D)).

As will be clear to the skilled person (for example on the basis of thefurther disclosure herein), affinity can be determined in a manner knownper se, depending on the specific antigen of interest. Avidity is themeasure of the strength of binding between an antigen-binding molecule(such as an antibody or an effective part thereof of the invention) andthe pertinent antigen. Avidity is related to both the affinity betweenan antigenic determinant and its antigen binding site on theantigen-binding molecule and the number of pertinent binding sitespresent on the antigen-binding molecule. Typically, antigen-bindingproteins (such as an antibody or an effective part thereof of theinvention) will bind to their antigen with a dissociation constant(K_(D)) of 10⁻⁵ to 10⁻¹² M or less, and preferably 10⁻⁷ to 10⁻¹² M orless and more preferably 10⁻⁸ to 10⁻¹² M (i.e. with an associationconstant (K_(A)) of 10⁵ to 10¹² M⁻¹ or more, and preferably 10⁷ to 10¹²liter/moles or more and more preferably 10⁸ to 10¹² M⁻¹). Any K_(D)value greater than 10⁴M (or any K_(A) value lower than 10⁴M⁻¹) isgenerally considered to indicate non-specific binding. Preferably, amonovalent immunoglobulin sequence of the invention will bind to thedesired antigen with an affinity less than 500 nM, preferably less than200 nM, more preferably less than 10 nM, such as less than 500 pM.Specific binding of an antigen-binding protein to an antigen orantigenic determinant can be determined in any suitable manner known perse, including, for example, Scatchard analysis and/or competitivebinding assays, such as radioimmunoassays (RIA), enzyme immunoassays(EIA) and sandwich competition assays, and the different variantsthereof known per se in the art, as well as the other techniquesmentioned herein.

The dissociation constant may be the actual or apparent dissociationconstant, as will be clear to the skilled person. Methods fordetermining the dissociation constant will be clear to the skilledperson, and for example include the techniques mentioned herein. In thisrespect, it will also be clear that it may not be possible to measuredissociation constants of more than 10⁻⁴ M or 10⁻³M (e.g., of 10⁻² M).Optionally, as will also be clear to the skilled person, the (actual orapparent) dissociation constant may be calculated on the basis of the(actual or apparent) association constant (K_(A)), by means of therelationship [K_(D)=1/K_(A)].

The affinity denotes the strength or stability of a molecularinteraction. The affinity is commonly given as by the K_(D), ordissociation constant, which has units of mole/liter (or M). Theaffinity can also be expressed as an association constant, K_(A), whichequals 1/K_(D) and has units of (liter/mole)⁻¹ (or M⁻¹). In the presentspecification, the stability of the interaction between two molecules(such as an amino acid sequence, Nanobody or polypeptide of theinvention and its intended target) will mainly be expressed in terms ofthe K_(D) value of their interaction; it being clear to the skilledperson that in view of the relation K_(A)=1/K_(D), specifying thestrength of molecular interaction by its K_(D) value can also be used tocalculate the corresponding K_(A) value. The K_(D) value characterizesthe strength of a molecular interaction also in a thermodynamic sense asit is related to the free energy (DG) of binding by the well knownrelation DG=RT·ln(K_(D)) (equivalently DG=−RT·ln(K_(A))), where R equalsthe gas constant, T equals the absolute temperature and ln denotes thenatural logarithm.

The K_(D) for biological interactions which are considered meaningful(e.g. specific) are typically in the range of 10⁻¹⁰ M (0.1 nM) to 10⁻⁵M(10000 nM). The stronger an interaction, the lower its K_(D).

In a preferred embodiment, the K_(D) of the antibody of the invention isbetween 10⁻¹² M and 10⁻⁵ M, preferably less than 10⁻⁶, preferably lessthan 10⁻⁷, preferably less than 10⁻⁸ M, preferably less than 10⁻⁹ M,more preferably less than 10⁻¹⁰ M, even more preferably less than 10⁻¹¹M, most preferably less than 10⁻¹²M.

The K_(D) can also be expressed as the ratio of the dissociation rateconstant of a complex, denoted as k_(off), to the rate of itsassociation, denoted k_(on) (so that K_(D)=k_(off)/k_(on) andK_(A)=k_(on)/k_(off)). The off-rate k_(off) has units s⁻¹ (where s isthe SI unit notation for second). The on-rate k_(on) has units M⁻¹ s⁻¹.The on-rate may vary between 10² M⁻¹ s⁻¹ to about 10⁷ M⁻¹ s⁻¹,approaching the diffusion-limited association rate constant forbimolecular interactions. The off-rate is related to the half-life of agiven molecular interaction by the relation t_(1/2)=ln(2)/k_(off). Theoff-rate may vary between 10⁻⁶ s⁻¹ (near irreversible complex with at_(1/2) of multiple days) to 1 s⁻¹ (t_(1/2)=0.69 s).

The affinity of a molecular interaction between two molecules can bemeasured via different techniques known per se, such as the well knownsurface plasmon resonance (SPR) biosensor technique (see for exampleOber et al., Intern Immunology, 13, 1551-1559, 2001) where one moleculeis immobilized on the biosensor chip and the other molecule is passedover the immobilized molecule under flow conditions yielding k_(on),k_(off) measurements and hence K_(D) (or K_(A)) values. This can forexample be performed using the well-known BIACORE instruments.

It will also be clear to the skilled person that the measured K_(D) maycorrespond to the apparent K_(D) if the measuring process somehowinfluences the intrinsic binding affinity of the implied molecules forexample by artefacts related to the coating on the biosensor of onemolecule. Also, an apparent K_(D) may be measured if one moleculecontains more than one recognition site for the other molecule. In suchsituation the measured affinity may be affected by the avidity of theinteraction by the two molecules.

Another approach that may be used to assess affinity is the 2-step ELISA(Enzyme-Linked Immunosorbent Assay) procedure of Friguet et al. (J.Immunol. Methods, 77, 305-19, 1985). This method establishes a solutionphase binding equilibrium measurement and avoids possible artefactsrelating to adsorption of one of the molecules on a support such asplastic.

However, the accurate measurement of K_(D) may be quite labor-intensive;therefore, apparent K_(D) values are often determined in order to assessthe binding strength of two molecules. It should be noted that as longas all measurements are made in a consistent way (e.g. keeping the assayconditions unchanged), apparent K_(D) measurements can be used as anapproximation of the true K_(D) and hence in the present document K_(D)and apparent K_(D) should be treated with equal importance or relevance.

Finally, it should be noted that in many situations the experiencedscientist may judge it to be convenient to determine the bindingaffinity relative to some reference molecule. For example, to assess thebinding strength between molecules A and B, one may e.g. use a referencemolecule C that is known to bind to B and that is suitably labelled witha fluorophore or chromophore group or other chemical moiety, such asbiotin for easy detection in ELISA or flow cytometry or other format(the fluorophore for fluorescence detection, the chromophore for lightabsorption detection, the biotin for streptavidin-mediated ELISAdetection). Typically, the reference molecule C is kept at a fixedconcentration and the concentration of A is varied for a givenconcentration or amount of B. As a result an Inhibitory Concentration(IC)₅₀ value is obtained corresponding to the concentration of A atwhich the signal measured for C in absence of A is halved. ProvidedK_(D ref), the K_(D) of the reference molecule, is known, as well as thetotal concentration c_(ref) of the reference molecule, the apparentK_(D) for the interaction A-B can be obtained from following formula:K_(D)=IC₅₀/(1+c_(ref)/K_(Dref)). Note that if c_(ref)<<K_(Dref),K_(D)≈IC₅₀. Provided the measurement of the IC₅₀ is performed in aconsistent way (e.g. keeping c_(ref) fixed) for the binders that arecompared, the strength or stability of a molecular interaction can beassessed by the IC₅₀ and this measurement is judged as equivalent toK_(D) or to apparent K_(D) throughout this text.

Another aspect of the invention relates to a hybridoma cell line, whichproduces an antibody as defined above.

The problem underlying the present invention is furthermore solved by amethod for producing an antibody as defined above, characterized by thefollowing steps:

-   -   a) initiating an immune response in a non-human animal by        administering a polypeptide as defined above to said animal,    -   b) removing an antibody containing body fluid from said animal,        and    -   c) producing the antibody by subjecting said antibody containing        body fluid to further purification steps.

The invention further relates to a method for producing an antibody asdefined above, characterized by the following steps:

-   -   a) initiating an immune response in a non-human animal by        administering a polypeptide as defined above to said animal,    -   b) removing the spleen or spleen cells from said animal,    -   c) producing hybridoma cells of said spleen or spleen cells,    -   d) selecting and cloning hybridoma cells specific for said        polypeptide,    -   e) producing the antibody by cultivation of said cloned        hybridoma cells, and    -   f) optionally conducting further purification steps.        Pharmaceutical Compositions and Related Medical Aspects

Another aspect of the present invention is related to a pharmaceuticalcomposition comprising

-   -   (i) the polypeptide according to the present invention, the        nucleic acid according to the present invention, and/or the        antibody according to the present invention; and    -   (ii) optionally a pharmaceutically acceptable excipient.

Accordingly, the polypeptide according to the present invention, thenucleic acid according to the present invention, the antibody accordingto the present invention or the pharmaceutical composition according tothe present invention may be

-   -   for use as a medicament, particularly as a vaccine or    -   for use in a method of treating or preventing a Borrelia        infection, particularly a B. burgdorferi s.s., B. garinii, B.        afzelii, B. andersoni, B. bavariensis, B. bissettii, B.        valaisiana, B. lusitaniae, B. spielmanii, B. japonica, B.        tanukii, B. turdi or B. sinica infection, preferably a B.        burgdorferi s.s., B. afzelii or B. garinii infection.

Preferably, the composition additionally comprises Lip-S1D1-S2D1 (SEQ IDNO: 29) and Lip-S5D1-S6D1 (SEQ ID NO: 33), the composition additionallycomprises Lip-S1D1-S2D1 (SEQ ID NO: 29), the composition additionallycomprises Lip-S5D1-S6D1 (SEQ ID NO: 33), or the composition comprisesLip-S1D1-S2D1 (SEQ ID NO: 29), Lip-S4D1-S3hybD1 (SEQ ID NO: 27) andLip-S5D1-S6D1 (SEQ ID NO: 33).

Still another aspect relates to a pharmaceutical composition as definedabove for use in the treatment or prevention of an infection withBorrelia species, more preferably pathogenic Borrelia species asdisclosed herein more preferably comprising B. burgdorferi s.s., B.afzelii, B. bavariensis and B. garinii.

The problem underlying the present invention is solved in another aspectby the use of the polypeptide according to the present invention, thenucleic acid according to the present invention, and/or the antibodyaccording to the present invention for the preparation of apharmaceutical composition for treating or preventing infections withBorrelia species, more preferably pathogenic Borrelia species asdisclosed herein more preferably comprising B. burgdorferi s.s., B.afzelii, B. bavariensis and B. garinii.

The pharmaceutical composition may optionally contain anypharmaceutically acceptable carrier or excipient, such as buffersubstances, stabilisers or further active ingredients, especiallyingredients known in connection with pharmaceutical compositions and/orvaccine production. Preferably, the pharmaceutically acceptableexcipient comprises L-methionine. Preferably, the pharmaceuticalcomposition is for use as a medicament, particularly as a vaccine or forpreventing or treating an infection caused by Borrelia species, morepreferably pathogenic Borrelia species as disclosed herein morepreferably comprising B. burgdorferi s.s., B. afzelii, B. bavariensisand B. garinii, and/or other pathogens against which the antigens havebeen included in the vaccine. Preferably, the pharmaceutical compositionis for use in a method of treating or preventing a Borrelia infection,particularly a B. burgdorferi s.s., B. garinii, B. afzelii, B.andersoni, B. bavariensis, B. bissettii, B. valaisiana, B. lusitaniae,B. spielmanii, B. japonica, B. tanukii, B. turdi or B. sinica infection,preferably a B. burgdorferi s.s., B. afzelii or B. garinii infection.

In one embodiment the pharmaceutical composition further comprises anadjuvant. The choice of a suitable adjuvant to be mixed with bacterialtoxins or conjugates made using the processes of the invention is withinthe knowledge of the person skilled in the art. Suitable adjuvantsinclude an aluminium salt such as aluminium hydroxide or aluminumphosphate, but may also be other metal salts such as those of calcium,magnesium, iron or zinc, or may be an insoluble suspension of acylatedtyrosine, or acylated sugars, cationically or anionically derivatizedsaccharides, or polyphosphazenes. In a preferred embodiment, thepharmaceutical composition is adjuvanted with aluminium hydroxide.

In a further embodiment, the pharmaceutical composition furthercomprises an immunostimulatory substance, preferably selected from thegroup consisting of polycationic polymers, especially polycationicpeptides, immunostimulatory oligodeoxynucleotides (ODNs), especiallyoligo(dIdC)₁₃ (SEQ ID NO: 63), peptides containing at least twoLysLeuLys motifs, especially peptide KLKLLLLLKLK (SEQ ID NO: 61),neuroactive compounds, especially human growth hormone, aluminiumhydroxide, aluminium phosphate, Freund's complete or incompleteadjuvants, or combinations thereof. Preferably, the immunostimulatorysubstance is a combination of either a polycationic polymer andimmunostimulatory deoxynucleotides or of a peptide containing at leasttwo LysLeuLys motifs and immunostimulatory deoxynucleotides, preferablya combination of KLKLLLLLKLK (SEQ ID NO: 61) and oligo(dIdC)₁₃ (SEQ IDNO: 63). More preferably, said polycationic peptide is polyarginine.

In a further embodiment, the pharmaceutical composition comprises sodiumphosphate, sodium chloride, L-methionine, sucrose and Polysorbate-20(Tween-20) at a pH of 6.7+/−0.2. Preferably, the pharmaceuticalcomposition also comprises aluminium hydroxide, preferably at aconcentration of 0.15%.

In one embodiment, the formulation comprises between 5 mM and 50 mMsodium phosphate, between 100 and 200 mM sodium chloride, between 5 mMand 25 mM L-Methionine, between 2.5% and 10% Sucrose, between 0.01% and0.1% Tween 20 and between 0.1% and 0.2% (w/v) aluminium hydroxide. Morepreferably, the formulation comprises 10 mM sodium phosphate, 150 mMsodium chloride, 10 mM L-Methionine, 5% Sucrose, 0.05% Tween 20 and0.15% (w/v) aluminium hydroxide at pH 6.7±0.2. Even more preferably, theformulation comprises at least one, at least two, at least three mutantOspA heterodimers according to the invention.

In one embodiment, the pharmaceutical composition comprises 3heterodimers, preferably Lip-S1D1-S2D1 (SEQ ID NO: 29), Lip-S4D1-S3hybD1(SEQ ID NO: 27) and Lip-S5D1-S6D1 (SEQ ID NO: 33). Preferably, the threeheterodimers are mixed at a molar ratio of 1:2:1, 1:3:1, 1:1:2, 1:1:3,1:2:2, 1:2:3, 1:3:2, 1:3:3, 2:1:1, 2:1:2, 2:1:3, 2:2:3, 2:2:1, 2:3:1,2:3:2, 2:3:3, 3:1:1, 3:1:2, 3:1:3, 3:2:1, 3:2:2, 3:2:3, 3:3:1, 3:3:2,most preferably 1:1:1.

In a further embodiment, the pharmaceutical composition comprises twoheterodimers, preferably Lip-S1D1-S2D1 (SEQ ID NO: 29) and Lip-S5D1-S6D1(SEQ ID NO: 33), Lip-S1D1-S2D1 (SEQ ID NO: 29) and Lip-S4D1-S3hybD1 (SEQID NO: 27) or Lip-S4D1-S3hybD1 (SEQ ID NO: 27) and Lip-S5D1-S6D1 (SEQ IDNO: 33) in a molar ratio of 1:2, 1:3, 2:1, 3:1, 2:3, 3:2, preferably1:1.

In one embodiment the pharmaceutical composition or vaccine of theinvention further comprises at least one additional antigen fromBorrelia or a pathogen other than Borrelia (herein referred togenerically as “combination pharmaceutical composition or vaccine”). Ina preferred embodiment, the at least one additional antigen is derivedfrom a Borrelia species causing Lyme borreliosis. In various aspects,the at least one additional antigen is derived from another pathogen,preferably a tick-borne pathogen. In a further aspect, the pathogencauses Rocky Mountain spotted fever, Human granulocytic ehrlichiosis(HGE), Sennetsu Fever, Human Monocytic Ehrlichiosis (HME), Anaplasmosis,Boutonneuse fever, Rickettsia parkeri Rickettsiosis, SouthernTick-Associated Rash Illness (STARI), Helvetica Spotted fever, 364DRickettsiosis, African spotted fever, Relapsing fever, Tularemia,Colorado tick fever, Tick-borne encephalitis (TBE, also known as FSME),Crimean-Congo hemorrhagic fever, Q fever, Omsk hemorrhagic fever,Kyasanur forest disease, Powassan encephalitis, Heartland virus diseaseor Babesiosis. In a further aspect, the disease is Japaneseencephalitis.

In a further embodiment, the at least one additional antigen is derivedfrom a vector-borne, preferably a tick-borne, pathogen selected from thegroup comprising Borrelia hermsii, Borrelia parkeri, Borrelia duttoni,Borrelia miyamotoi, Borrelia turicatae, Rickettsia rickettsii,Rickettsia australis, Rickettsia conori, Rickettsia helvetica,Francisella tularensis, Anaplasma phagocytophilum, Ehrlichia sennetsu,Ehrlichia chaffeensis, Neoehrlichia mikurensis, Coxiella burnetii andBorrelia lonestari, Tick-borne encephalitis virus (TBEV aka FSME virus),Colorado tick fever virus (CTFV), Crimean-Congo hemorrhagic fever virus(CCHFV), Omsk Hemorrhagic Fever virus (OHFV), Japanese encepalitis virus(JEV) and Babesia spp.

In another aspect, the invention relates to a kit comprising thepharmaceutical composition of the present invention and an additionalantigen as defined above, wherein the at least one additional antigen iscomprised in a second composition, particularly wherein the secondcomposition is a vaccine, preferably a tick-borne encephalitis vaccine,a Japanese encephalitis vaccine or a Rocky Mountain spotted fevervaccine. The first composition may be a vaccine as well. The combinationpharmaceutical composition or vaccine of the invention comprises anycomposition discussed herein in combination with at least a second(vaccine) composition. In some aspects, the second vaccine compositionprotects against a vector-borne disease, preferably a tick-bornedisease. In various aspects, the second vaccine composition has aseasonal immunization schedule compatible with immunization againstBorrelia infection or Lyme borreliosis. In other aspects, combinationvaccines are useful in the prevention of multiple diseases for use ingeographical locations where these diseases are prevalent.

In one aspect, the second composition is a vaccine selected from thegroup consisting of a tick-borne encephalitis vaccine, a Japaneseencephalitis vaccine, and a Rocky Mountain Spotted Fever vaccine. In apreferred aspect, the vaccine composition is FSME-IMMUN® (Baxter),Encepur® (Novartis Vaccines), EnceVir® (Microgen NPO) or TBE MoscowVaccine® (Chumakov Institute of Poliomyelitis and Viral Encephalitidesof Russian Academy of Medical Sciences). In another preferred aspect,the vaccine composition is IXIARO®/JESPECT® (Valneva SE), JEEV®(Biological E, Ltd.) or IMOJEV® (Sanofi Pasteur).

There is further provided a pharmaceutical composition which is avaccine, this vaccine may further comprise a pharmaceutically acceptableexcipient. In a preferred embodiment, the excipient is L-methionine.

The invention also includes immunogenic compositions. In some aspects,an immunogenic composition of the invention comprises any of thecompositions discussed herein and a pharmaceutically acceptable carrier.In various aspects, the immunogenic composition has the property ofinducing production of an antibody that specifically binds an outersurface protein A (OspA) protein. In certain aspects, the immunogeniccomposition has the property of inducing production of an antibody thatspecifically binds Borrelia. In particular aspects, the immunogeniccomposition has the property of inducing production of an antibody thatneutralizes Borrelia. In some aspects, the antibody is produced by ananimal. In further aspects, the animal is a mammal. In even furtheraspects, the mammal is human.

The vaccine preparations containing pharmaceutical compositions of thepresent invention may be used to protect a mammal susceptible toBorrelia infection or treat a mammal with a Borrelia infection, by meansof administering said vaccine via a systemic or mucosal route. Theseadministrations may include injection via the intramuscular,intraperitoneal, intradermal or subcutaneous routes; or via mucosaladministration to the oral/alimentary, respiratory or genitourinarytracts. Although the vaccine of the invention may be administered as asingle dose, components thereof may also be co-administered together atthe same time or at different times.

In one aspect of the invention is provided a vaccine kit, comprising avial containing a pharmaceutical composition of the invention,optionally in lyophilised form, and further comprising a vial containingan adjuvant as described herein. It is envisioned that in this aspect ofthe invention, the adjuvant will be used to reconstitute the lyophilisedimmunogenic composition. In a further aspect, the pharmaceuticalcomposition of the invention may be pre-mixed in a vial, preferably in asyringe.

A further aspect of the invention is a method of treating or preventinga Borrelia infection in a subject in need thereof comprising the step ofadministering to the subject a therapeutically-effective amount of apolypeptide of the present invention, the nucleic acid of the presentinvention, the antibody of the present invention or the pharmaceuticalcomposition of the present invention.

A further aspect of the invention is a method of immunizing a subject inneed thereof comprising the step of administering to the subject atherapeutically-effective amount of a polypeptide of the presentinvention, the nucleic acid of the present invention, the antibody ofthe present invention or the pharmaceutical composition of the presentinvention.

A still further aspect relates to a method for immunizing an animal orhuman against infection with a Borrelia organism, comprising the step ofadministering to said animal or human an effective amount a polypeptideof the present invention, the nucleic acid of the present invention, theantibody of the present invention or the pharmaceutical composition ofthe present invention, wherein the effective amount is suitable toelicit an immune response in said animal or human.

Yet another aspect relates to a method for stimulating an immuneresponse in an animal or human against a Borrelia organism, comprisingthe step of administering to said animal or human an effective amount ofa polypeptide of the present invention, the nucleic acid of the presentinvention, the antibody of the present invention or the pharmaceuticalcomposition of the present invention, wherein the effective amount issuitable to stimulate an immune response in said animal or human.

Preferably, the Borrelia organism is selected from the group comprisingB. burgdorferi, particularly B. burgdorferi s.s., B. garinii, B.afzelii, B. andersoni, B. bavariensis, B. bissettii, B. valaisiana, B.lusitaniae, B. spielmanii, B. japonica, B. tanukii, B. turdi or B.sinica, preferably B. burgdorferi s.s., B. afzelii or B. garinii.

In one embodiment there is provided a method of preventing or treatingprimary and/or recurring episodes of Borrelia infection comprisingadministering to the host an immunoprotective dose of the pharmaceuticalcomposition or vaccine or kit of the invention.

A further aspect of the invention is a pharmaceutical composition of theinvention for use in the treatment or prevention of Borrelial disease.In one embodiment there is provided a pharmaceutical composition for usein the treatment or prevention of Borrelia infection.

A further aspect of the invention is the use of the pharmaceuticalcomposition or vaccine or kit of the invention in the manufacture of amedicament for the treatment or prevention of Borrelia infection. In oneembodiment there is provided a pharmaceutical composition of theinvention for use in the manufacture of a medicament for the treatmentor prevention of Borrelia infection.

The invention also includes methods for inducing an immunologicalresponse in a subject. In various aspects, such methods comprise thestep of administering any of the immunogenic compositions or vaccinecompositions discussed herein to the subject in an amount effective toinduce an immunological response. In certain aspects, the immunologicalresponse comprises production of an anti-OspA antibody.

The invention includes methods for preventing or treating a Borreliainfection or Lyme boreliosis in a subject. In various aspects, suchmethods comprise the step of administering any of the vaccinecompositions discussed herein or any of the combination vaccinesdiscussed herein to the subject in an amount effective to prevent ortreat the Borrelia infection or Lyme borreliosis.

The invention includes uses of polypeptides, nucleic acids, antibodies,pharmaceutical compositions or vaccines of the invention for thepreparation of medicaments. Other related aspects are also provided inthe instant invention.

The terms “comprising”, “comprise” and “comprises” herein are intendedby the inventors to be optionally substitutable with the terms“consisting of”, “consist of” and “consists of”, respectively, in everyinstance. The term “comprises” means “includes”. Thus, unless thecontext requires otherwise, the word “comprises”, and variations such as“comprise” and “comprising” will be understood to imply the inclusion ofa stated compound or composition (e.g., nucleic acid, polypeptide,antibody) or step, or group of compounds or steps, but not to theexclusion of any other compounds, composition, steps, or groups thereof.The abbreviation, “e.g.” is derived from the Latin exempli gratia, andis used herein to indicate a non-limiting example. Thus, theabbreviation “e.g.” is synonymous with the term “for example”.

Embodiments herein relating to “vaccine compositions” of the inventionare also applicable to embodiments relating to “pharmaceuticalcompositions” of the invention, and vice versa.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. Definitions of commonterms in molecular biology can be found in Benjamin Lewin, Genes V,published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrewet al. (eds.), The Encyclopedia of Molecular Biology, published byBlackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers(ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

The singular terms “a”, “an”, and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. The term “plurality” refers to two or more. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values given for nucleic acids or polypeptidesare approximate, and are provided for description. Additionally,numerical limitations given with respect to concentrations or levels ofa substance, such as an antigen, may be approximate.

A preferable carrier or excipient for the polypeptides according to thepresent invention in their diverse embodiments, or a nucleic acidmolecule according to the present invention is an immunostimulatorycompound such as an adjuvant for further stimulating the immune responseto the polypeptide according to the present invention or a codingnucleic acid molecule thereof.

Adjuvants or immunostimulatory compounds may be used in compositions ofthe invention. Preferably, the immunostimulatory compound inpharmaceutical compositions according to the present invention isselected from the group of polycationic substances, especiallypolycationic peptides, immunostimulatory nucleic acids molecules,preferably immunostimulatory deoxynucleotides, oil-in-water orwater-in-oil emulsions, MF59, aluminium salts, Freund's completeadjuvant, Freund's incomplete adjuvant, neuroactive compounds,especially human growth hormone, or combinations thereof.

The use of an aluminium hydroxide and/or aluminium phosphate adjuvant isparticularly preferred, and antigens are generally adsorbed to thesesalts. Preferably, aluminium hydroxide is present at a finalconcentration of 0.15%. A useful aluminium phosphate adjuvant isamorphous aluminium hydroxyphosphate with PO₄/A1 molar ratio between0.84 and 0.92. Another adjuvant useful in the current invention is analuminium salt that is able to provide an aqueous composition havingless than 350 ppb heavy metal based on the weight of the aqueouscomposition. A further useful aluminium-based adjuvant is ASO4, acombination of aluminium hydroxide and monophosphoryl lipid A (MPL).

Also, the pharmaceutical composition in accordance with the presentinvention is a pharmaceutical composition which comprises at least anyof the following compounds or combinations thereof: the nucleic acidmolecules according to the present invention, the polypeptides accordingto the present invention in their diverse embodiments, the vectoraccording to the present invention, the cells according to the presentinvention and the antibody according to the present invention. Inconnection therewith, any of these compounds may be employed incombination with a non-sterile or sterile carrier or carriers for usewith cells, tissues or organisms, such as a pharmaceutical carriersuitable for administration to a subject. Such carriers may include, butare not limited to, saline, buffered saline, dextrose, water, glycerol,ethanol and combinations thereof. The formulation should suit the modeof administration.

In one embodiment, the pharmaceutical composition comprises astabilizer. The term “stabilizer” refers to a substance or vaccineexcipient which protects the immunogenic composition of the vaccine fromadverse conditions, such as those which occur during heating orfreezing, and/or prolongs the stability or shelf-life of the immunogeniccomposition in a stable and immunogenic condition or state. Examples ofstabilizers include, but are not limited to, sugars, such as sucrose,lactose and mannose; sugar alcohols, such as manitol; amino acids, suchas glycine or glutamic acid; and proteins, such as human serum albuminor gelatin.

The pharmaceutical compositions of the present invention may beadministered in any effective, convenient manner including, forinstance, administration by topical, oral, anal, vaginal, intravenous,intraperitoneal, intramuscular, subcutaneous, intranasal, intratrachealor intradermal routes, among others. In a preferred embodiment, thepharmaceutical compositions are administered subcutaneously orintramuscularly, most preferably intramuscularly.

In therapy or as a prophylactic, the active agent of the pharmaceuticalcomposition of the present invention may be administered to anindividual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

Alternatively the composition, preferably the pharmaceutical compositionmay be formulated for topical application, for example in the form ofointments, creams, lotions, eye ointments, eye drops, ear drops,mouthwash, impregnated dressings and sutures and aerosols, and maycontain appropriate conventional additives, including, for example,preservatives, solvents to assist drug penetration, and emollients inointments and creams Such topical formulations may also containcompatible conventional carriers, for example cream or ointment bases,and ethanol or oleyl alcohol for lotions. Such carriers may constitutefrom about 1% to about 98% by weight of the formulation; more usuallythey will constitute up to about 80% by weight of the formulation.

In addition to the therapy described above, the compositions of thisinvention may be used generally as a wound treatment agent to preventadhesion of bacteria to matrix proteins exposed in wound tissue and forprophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

In a preferred embodiment the pharmaceutical composition is a vaccinecomposition. Preferably, such vaccine composition is conveniently ininjectable form. Conventional adjuvants may be employed to enhance theimmune response. A suitable unit dose for vaccination with a proteinantigen is for adults between 0.02 μg and 3 μg antigen per kg bodyweight and for children between 0.2 μg and 10 μg antigen per kg bodyweight, and such dose is preferably administered 1 to 3 times atintervals of 2 to 24 weeks.

At the indicated dose range, no adverse toxicological effects areexpected with the compounds of the invention, which would preclude theiradministration to suitable individuals.

As an additional aspect, the invention includes kits which comprise oneor more pharmaceutical formulations for administration to a subjectpackaged in a manner which facilitates their use for administration tosubjects. In a preferred embodiment, the kits comprise the formulationin a final volume of 2 mL, more preferably in a final volume of 1 mL.

In a specific embodiment, the invention includes kits for producing asingle dose administration unit. The kits, in various aspects, eachcontain both a first container having a dried protein and a secondcontainer having an aqueous formulation. Also included within the scopeof this invention are kits containing single and multi-chamberedpre-filled syringes (e.g., liquid syringes and lyosyringes).

In another embodiment, such a kit includes a pharmaceutical formulationdescribed herein (e.g., a composition comprising a therapeutic proteinor peptide), packaged in a container such as a sealed bottle or vessel,with a label affixed to the container or included in the package thatdescribes use of the compound or composition in practicing the method.In one embodiment, the pharmaceutical formulation is packaged in thecontainer such that the amount of headspace in the container (e.g., theamount of air between the liquid formulation and the top of thecontainer) is very small. Preferably, the amount of headspace isnegligible (i.e., almost none).

In one aspect, the kit contains a first container having a therapeuticprotein or peptide composition and a second container having aphysiologically acceptable reconstitution solution for the composition.In one aspect, the pharmaceutical formulation is packaged in a unitdosage form. The kit optionally further includes a device suitable foradministering the pharmaceutical formulation according to a specificroute of administration. In some aspects, the kit contains a label thatdescribes use of the pharmaceutical formulations.

The pharmaceutical composition can contain a range of differentantigens. Examples of antigens are whole-killed or attenuated organisms,subfractions of these organisms, proteins, or, in their most simpleform, peptides. Antigens can also be recognized by the immune system inthe form of glycosylated proteins or peptides and may also be or containpolysaccharides or lipids. Short peptides can be used, since cytotoxic Tcells (CTL) recognize antigens in the form of short, usually 8-11 aminoacid long, peptides in conjunction with major histocompatibility complex(MHC). B cells can recognize linear epitopes as short as 4 to 5 aminoacids, as well as three-dimensional structures (conformationalepitopes).

In a preferred embodiment, the pharmaceutical composition of anotheraspect additionally comprises a hyperimmune serum-reactive antigenagainst a Borrelia protein or an active fragment or variant thereof,such as, e.g., the antigens, fragments and variants as described inWO2008/031133.

According to the invention, the pharmaceutical composition according toanother aspect may be used as a medicament, particularly as a vaccine,particularly in connection with a disease or disease condition which iscaused by, linked or associated with Borrelia.

The pharmaceutical composition of the present invention may be used as amedicament, particularly as a vaccine, particularly in connection with adisease or disease condition which is caused by, linked with orassociated with Borrelia, more preferably any pathogenic Borreliaspecies and more preferably in a method for treating or preventing aBorrelia infection, particularly a B. burgdorferi s.s., B. garinii, B.afzelii, B. andersoni, B. bavariensis, B. bissettii, B. valaisiana, B.lusitaniae, B. spielmanii, B. japonica, B. tanukii, B. turdi or B.sinica infection, preferably a B. burgdorferi s.s., B. afzelii or B.garinii infection.

In connection therewith, it should be noted that the various Borreliaspecies, including B. burgdorferi s.l., comprise several species andstrains including those disclosed herein. A disease related, caused orassociated with the bacterial infection to be prevented and/or treatedaccording to the present invention includes Lyme borreliosis (Lymedisease). Further aspects, symptoms, stages and subgroups of Lymeborreliosis as well as specific groups of patients suffering from suchdisease as also disclosed herein, including in the introductory part,are incorporated herein by reference. More specifically, Lymeborreliosis generally occurs in stages, with remission and exacerbationswith different clinical manifestation at each stage. Early infectionstage 1 consists of localized infection of the skin, followed withindays or weeks by stage 2, disseminated infection, and months to yearslater by stage 3, persistent infection. However, the infection isvariable; some patients have only localized infections of the skin,while others display only later manifestations of the illness, such asarthritis.

In a fourth aspect, the present invention relates to a method oftreating or preventing a Borrelia infection in a subject in needthereof, comprising the step of administering to the subject atherapeutically effective amount of a pharmaceutical compositionaccording to the third aspect.

The term “subject” is used throughout the specification to describe ananimal, preferably a mammal, more preferably a human, to whom atreatment or a method according to the present invention is provided.For treatment of those infections, conditions or disease states whichare specific for a specific animal such as a human patient, the termpatient refers to that specific animal. Preferably, the subject is ahuman; however, the medical use of the composition may also includeanimals such as poultry including chicken, turkey, duck or goose,livestock such as horse, cow or sheep, or companion animals such as dogsor cats.

The term “effective amount” is used throughout the specification todescribe an amount of the present pharmaceutical composition which maybe used to induce an intended result when used in the method of thepresent invention. In numerous aspects of the present invention, theterm effective amount is used in conjunction with the treatment orprevention. In other aspects, the term effective amount simply refers toan amount of an agent which produces a result which is seen as beingbeneficial or useful, including in methods according to the presentinvention where the treatment or prevention of a Borrelia infection issought.

The term effective amount with respect to the presently describedcompounds and compositions is used throughout the specification todescribe that amount of the compound according to the present inventionwhich is administered to a mammalian patient, especially including ahuman patient, suffering from a Borrelia-associated disease, to reduceor inhibit a Borrelia infection.

In a preferred embodiment, the method of immunizing a subject accordingto the above aspect comprises the step of administering to the subject atherapeutically effective amount of a pharmaceutical composition of thethird aspect of the current invention.

The method comprises inducing an immunological response in an individualthrough gene therapy or otherwise, by administering a polypeptide ornucleic acid according to the present invention in vivo in order tostimulate an immunological response to produce antibodies or acell-mediated T cell response, either cytokine-producing T cells orcytotoxic T cells, to protect said individual from disease, whether ornot that disease is already established within the individual.

The products of the present invention, particularly the polypeptides andnucleic acids, are preferably provided in isolated form, and may bepurified to homogeneity. The term “isolated” as used herein meansseparated “by the hand of man” from its natural state; i.e., if itoccurs in nature, it has been changed or removed from its originalenvironment, or both. For example, a naturally-occurring nucleic acidmolecule or a polypeptide naturally present in a living organism in itsnatural state is not “isolated”, but the same nucleic acid molecule orpolypeptide separated from the coexisting materials of its natural stateis “isolated”, as the term is employed herein. As part of or followingisolation, such nucleic acid molecules can be joined to other nucleicacid molecules, such as DNA molecules, for mutagenesis, to form fusiongenes, and for propagation or expression in a host, for instance. Theisolated nucleic acid molecules, alone or joined to other nucleic acidmolecules such as vectors, can be introduced into host cells, in cultureor in whole organisms. Introduced into host cells in culture or in wholeorganisms, such DNA molecules still would be isolated, as the term isused herein, because they would not be in their naturally-occurring formor environment. Similarly, the nucleic acid molecules and polypeptidesmay occur in a composition, such as medium formulations, solutions forintroduction of nucleic acid molecules or polypeptides, for example,into cells, compositions or solutions for chemical or enzymaticreactions, for instance, which are not naturally occurring compositions,and, therein remain isolated nucleic acid molecules or polypeptideswithin the meaning of that term as it is employed herein.

The invention is not limited to the particular methodology, protocolsand reagents described herein because they may vary. Furthermore, theterminology used herein is for the purpose of describing particularembodiments only and is not intended to limit the scope of the presentinvention. As used herein and in the appended claims, the singular forms“a”, “an”, and “the” include plural reference unless the context clearlydictates otherwise. Similarly, the words “comprise”, “contain” and“encompass” are to be interpreted inclusively rather than exclusively.

Unless defined otherwise, all technical and scientific terms and anyacronyms used herein have the same meanings as commonly understood byone of ordinary skill in the art in the field of the invention. Althoughany methods and materials similar or equivalent to those describedherein can be used in the practice of the present invention, thepreferred methods, and materials are described herein.

The present invention is further illustrated by the following Figures,Tables, Examples and the Sequence listing, from which further features,embodiments and advantages may be taken. As such, the specificmodifications discussed are not to be construed as limitations on thescope of the invention. It will be apparent to the person skilled in theart that various equivalents, changes, and modifications may be madewithout departing from the scope of the invention, and it is thus to beunderstood that such equivalent embodiments are to be included herein.

In connection with the present invention

FIG. 1 shows electrostatic potential isocontours ofdisulfide-bond-stabilized fragments of serotype 1-serotype 6 and B.valaisiana OspA (S1D1-S6D1 and BvaD1), as well as the mutant fusion OspAfragment of the invention consisting of amino acids 125-176 of B.valaisiana, strain VS116, and amino acids 177-274 of B. garinii, strainPBr, with an introduced disulfide bond (S3hybD1).

FIG. 2 shows the amino acid alignment of Lip-S4D1-S3hybD1 (SEQ ID NO:27), the mutant serotype 3 OspA fusion fragment-containing heterodimerprotein of the invention, with the heterodimer protein Lip-S4D1-S3D1(SEQ ID NO: 31).

FIG. 3 schematically shows the production of mutant OspA fragmentheterodimers according to the current invention.

FIG. 4 schematically represents the polypeptide components of apharmaceutical composition of the current invention, an “improvedcombination vaccine”, comprising three different mutant OspAheterodimers, including Lip-S4D1-S3hybD1.

FIG. 5 shows the chemical structure of Pam₃Cys, an example of a fattyacid substituted cysteine, such as would be found at the N-terminus oflipidated polypeptides of the current invention.

FIG. 6 shows IgG antibody titers to Borrelia OspA proteins of serotype1-6 produced in mice in response to immunization with the improvedheterodimer combination vaccine of the invention.

FIG. 7 shows the binding of antibodies from mice immunized with theimproved heterodimer combination vaccine of the invention to the cellsurface of Borrelia spirochetes of OspA serotypes 1-6.

Table 1 compares the purification yield of the Lip-S4D1-S3hybD1heterodimer and the Lip-S4D1-S3D1 heterodimer.

Table 2 shows the protective capacity of the improved heterodimercombination vaccine of the invention against in vivo challenge with OspAserotypes 1, 2, 5 and 6 Borrelia.

The figures and tables which may be referred to in the specification aredescribed below in more detail.

FIG. 1 Electrostatic potential simulation of thedisulfide-bond-stabilized OspA fragments from serotypes 1-6 (S1D1-S6D1)and B. valaisiana (BvaD1) as well as the disulfide-bond-stabilizedfusion OspA fragment (S3hybD1). Isocontours (+/−1 kT/e) are coloredbright for negative charges and dark for positive charges, as a solidsurface. The solvent-accessible surface is rendered as a wireframe. Thewhite arrows indicate the position of the two extended clusters givingrise to electrostatic polarity on the same plane of the serotype 3fragment. It can be seen that the surfaces of the hybrid OspA C-terminalfragment do not possess the extended electrostatic polar clusters as theS3D1 monomer.

FIG. 2 Amino acid sequence alignment of Lip-S4D1-S3hybD1 (SEQ ID NO: 27)and Lip-S4D1-S3D1 (SEQ ID NO: 31) heterodimer polypeptides, showing theconsensus sequence. The Lip-S4D1-S3hybD1 heterodimer differs from theLip-S4D1-S3D1 heterodimer by only 31 amino acids in total.

FIG. 3 Production of a mutant OspA heterodimer of the inventioncomprising two mutant OspA C-terminal fragments selected from differentOspA serotypes of Borrelia sp. or a hybrid mutant OspA C-terminalfragment (A) Schematic representation of a nucleic acid encoding alipidated mutant OspA heterodimer. The components, from 5′ to 3′,comprise the coding sequences for a lipidation signal sequence (Lipsignal), a CSS peptide for N-terminal lipidation, a mutant C-terminalfragment of OspA with two non-native cysteines, a short linker peptide(LN1), followed by a second mutant OspA C-terminal fragment with twonon-native cysteines. (B) The intermediate mutant OspA heterodimerpolypeptide comprises the nascent product directly following translationof the nucleic acid construct. From the N- to the C-terminus, thispolypeptide consists of a lipidation signal sequence (Lip signal), a CSSpeptide for lipidation, a mutant OspA fragment with a non-nativedisulfide bond, a short linker peptide (LN1), followed by a secondmutant OspA fragment with a non-native disulfide bond. (C) The finallipidated mutant OspA heterodimer polypeptide after post-translationalmodification. The heterodimer, from the N- to the C-terminus, consistsof a CSS peptide with the N-terminal cysteine lipidated, a mutant OspAfragment stabilized by a disulfide bond, a linker peptide (LN1), and asecond mutant OspA fragment stabilized by a disulfide bond. Thelipidation signal sequence is cleaved off during post-translationalmodification of the polypeptide as shown.

FIG. 4 An example of a preferred pharmaceutical composition according tothe current invention. Three mutant OspA heterodimers, each comprisingtwo mutated OspA fragments selected from different Borrelia OspAserotypes or a hybrid mutant OspA C-terminal fragment (S3hybD1) arepresent in the composition, together providing OspA antigens from sixdifferent Borrelia OspA serotypes. Such a pharmaceutical compositionenables simultaneous immunization against six Borrelia serotypes.

FIG. 5 Illustration of the chemical structure of Pam₃Cys, an example ofa fatty acid substitution of the N-terminal cysteine of full-lengthwild-type OspA protein as well as of lipidated mutant OspA fragmentheterodimers of the invention. During post-translational modification ofa full-length OspA protein or polypeptides of the invention, theN-terminal lipidation signal sequence is cleaved off and fatty acids,most commonly three palmitoyl moieties (“Pam₃”), are enzymaticallycovalently attached to the N-terminal cysteine residue (the sulfur atom,“S”, is indicated by an arrow). The remaining residues of thepolypeptide chain, which are located C-terminally from the Pam₃Cysresidue, are represented by “Xn”. (Modified from Bouchon, et al. (1997)Analytical Biochemistry 246: 52-61.)

FIG. 6 Antibody titers generated to all six serotypes of full-lengthOspA proteins. Mice were immunized three times with 3 μg each of theindicated combination vaccines: Lip-S1D1-S2D1, Lip-S4D1-S3D1 andLip-S5D1-S6D1 together in a 1:1:1 ratio (“Het combo”); Lip-S1D1-S2D1,Lip-54D1-S3hybD1 and Lip-S5D1-S6D1 together in a 1:1:1 ratio (“Improvedhet combo”) or with Lip-Chimeric OspA ST1/ST2-His, Lip-Chimeric OspAST5/ST3-His and Lip-Chimeric OspA ST6/ST4-His together in a 1:1:1 ratio(“Chimera combo”) at two week intervals and sera were collected at oneweek after the last dose. Titers of IgG antibodies to six differentserotypes of full-length OspA proteins were determined by ELISA.

FIG. 7 Binding of antibodies from immunized mice to the cell surface ofBorrelia spirochetes. Mice were immunized as above and sera werecollected at one week after the last dose and pooled. Serial dilutionsof the sera were tested for binding to the cell surface of Borreliaspirochetes via cell staining and flow cytometry. Fluorescence intensityvalues observed when staining with sera collected from control miceimmunized with Al(OH)₃ adjuvant alone were subtracted to account fornon-specific binding. (Borrelia used in the binding assay were: B.burgdorferi, OspA serotype 1, strain N40; B. afzelii, OspA serotype 2,strain PKo; B. garinii, OspA serotype 3, strain Fr; B. bavariensis, OspAserotype 4, strain Fin; B. garinii, OspA serotype 5, strain PHei; B.garinii, OspA serotype 6, strain KL11.)

EXAMPLES Example 1. Molecular Modelling of the Hybrid Serotype 3 OspAC-Terminal Fragment

Motivation to Construct Hybrid OspA ST3 C-Terminal Fragments

The Lyme borreliosis combination vaccine as described in our previousapplication (WO2014/006226) is composed of three mutant OspAheterodimers. Short stabilized fragments from two different OspAserotypes (ST), derived from the C-terminal domain, are fused with ashort linker to form a heterodimer. The three heterodimers are composedof OspA ST1-ST2, ST4-ST3 and ST5-ST6. For improvement of theimmunogenicity of the heterodimers, a signal sequence for lipidation isadded in analogy with mature full-length OspA which is a lipoprotein.

The lipidated heterodimer composed of OspA ST4-ST3 (Lip-S4D1-S3D1; SEQID NO: 31) proved to be less soluble than the two other heterodimers,which results in low recovery during purification. This problem canmostly be attributed to the short stabilized OspA ST3 portion since thisprotein, as a lipidated monomer, cannot be expressed and purified.

Molecular Modelling

A comparative structural investigation of the stabilized monomers wasuntertaken in silico to elucidate the compatibility of the folds betweenthe monomer models compared to the OspA crystal structure (PDB:1OSP; LiH, Dunn J J, Luft B J, Lawson C L (1997) Crystal structure of Lymedisease antigen outer surface protein A complexed with an Fab. Proc NatlAcad Sci 94: 3584-3589) and to compare their surface properties with theST3 type monomer. The crystal structure represents serotype 1 ofBorrelia burgdorferi B31, and shows OspA bound with its N-terminus, tothe murine antibody Fab 184.1. Homology structure models of the sixshort stabilized OspA monomers were constructed starting with the ST1OspA crystal structure and available homology models (SwissModel; KieferF, Arnold K, Kunzli M, Bordoli L, Schwede T (2009) The SWISS-MODELRepository and associated resources. Nucleic Acids Res 37: D387-392),which were then modified to incorporate the stabilizing disulfide bondsand sequence changes where applicable (The PyMOL Molecular GraphicsSystem, Version Open-Source, Schrödinger LLC).

The electrostatic potential isocontours of all six short stabilized OspAmonomers were simulated with the adaptive Poisson-Boltzmann solver(APBS; Baker N A, Sept D, Joseph S, Holst M J, McCammon J A (2001)Electrostatics of nanosystems: application to microtubules and theribosome. Proc Natl Acad Sci 98: 10037-10041, pdb2pqr; Dolinsky T J,Czodrowski P, Li H, Nielsen J E, Jensen J H, et al. (2007) PDB2PQR:expanding and upgrading automated preparation of biomolecular structuresfor molecular simulations. Nucleic Acids Res 35: W522-525) to determineif the short stabilized OspA ST3 protein has a pattern in surfaceelectrostatics which deviates from the other OspA STs, which couldexplain the solubility problem of short stabilized OspA ST3 (“S3D1”,FIG. 1 ). A significant polarity in the charge distribution was observedon one side of S3D1 (see arrows), which was not observed in any of theother short stabilized OspA STs.

An electrostatic potential simulation was also performed with shortstabilized OspA from B. valaisiana (“BvaD1”, FIG. 1 ). The BvaD1 OspAfragment displayed a variant polarity of the electrostatic potentialisocontours on the surface, which made it a potential candidate buildingblock for a partial segmental exchange with the aim to modify theoverall surface to not show any significant extended clusters ofextended electrostatic polarity as found in S3D1. The preferred linkbetween the serotype 3 part and the exchanged part from B. valaisiana inthe hybrid monomer was chosen to replace the N-terminal beta-sheet ofthe monomer and to leave two beta sheets and the C-terminal helix of theserotype 3 portion intact, under retention of the overall fold. Thelatter condition depends largely on the steric compatibility of thedensely packed hydrophobic residues in the core of the molecule. Foldcompatibility for the model of the hybrid stabilized monomer, S3hybD1,was verified with molecular mechanics simulation (Gromacs; Pronk S, PallS, Schulz R, Larsson P, Bjelkmar P, et al. (2013) GROMACS 4.5: ahigh-throughput and highly parallel open source molecular simulationtoolkit. Bioinformatics 29: 845-854).

Application in the Form of Heterodimers Containing the Hybrid OspAFragment

As a result of the electrostatic potential simulations, a newheterodimer containing the experimental fusion of fragments OspAproteins from B. valaisiana and B. garinii (S3hybD1, FIG. 1 ), wascloned: Lip-S4D1-S3hybD1. In addition to the changes of the firstone-third of the serotype 3 OspA fragment in the heterodimer,54D1-S3hybD1, compared with Lip S4D1-S3D1, an amino acid substitution atposition 233 (P233T, amino acid nomenclature according the immaturefull-length OspA; SEQ ID NO: 8) of OspA ST3 was introduced.

As illustrated in further detail below, this new heterodimer showssubstantially improved solubility as well as immunogenicity againstBorrelia expressing serotype 3 OspA when purified and used to immunizemice.

Example 2. Purification and Formulation of Lipidated Mutant OspAFragment Heterodimers

Cloning and Expression of Lipidated Non-His-Tagged Mutant OspA FragmentHeterodimers The fusion OspA monomer B. valaisiana/B. garinii strainVS116/PBr was codon-optimized for E. coli expression by GeneArt(Germany) The lipidation signal sequence added to the N-terminal end wasderived from the E. coli major outer membrane lipoprotein, Lpp, and wasfollowed directly C-terminally by a CSS peptide to provide an N-terminalcysteine for lipidation. The improved heterodimer construct wasgenerated by fusing the mutant serotype 4 OspA fragment and the hybridserotype 3 OspA fragment via the linker sequence “LN1”. Gene fragmentswere cloned into the pET28b(+) vector (Novagen, USA), and the stabilizedheterodimers were expressed in BL21(DE3) cells (Invitrogen, USA). Cellswere collected after 4 h by centrifugation and the pellet was stored at−70° C. for up to 12 months prior to further processing.Purification of Lipidated Mutant OspA Fragment Heterodimers

Cells were disrupted mechanically by high-pressure homogenization andthe lipidated mutant OspA fragment heterodimers, Lip-S4D1-S3D1 andLip-S4D1-S3hybD1, were enriched in the lipid phase by phase separation,using Triton X-114 as detergent. Subsequently, the diluted detergentphase was subjected to anion exchange chromatography (Q-sepharose; GEHealthcare, United Kingdom) operated in non-binding mode. The resultingflow-through was loaded on a hydroxyapatite column (Bio-Rad, USA) andthe lipidated proteins were eluted from the column by a linear saltgradient. The eluate was subjected to further purification over aDEAE-Sepharose column (GE Healthcare) in non-binding mode followed bygel filtration column (Superdex 200, GE Healthcare) for buffer exchange.The lipidated mutant OspA heterodimer peaks were pooled on the basis ofthe analytical size exclusion column and SDS-PAGE. After sterilefiltration, the purified heterodimers were stored at −20° C. untilformulation.

Formulation of the Combination Vaccine

Studies regarding the formulation of the combination vaccine of theinvention were carried out in order to optimize stability. Differenttypes of buffers and stabilizers were tested at various concentrationsin combination with aluminium hydroxide and antigen, as described in ourprevious application WO2014/006226. An optimal formulation of 40 μg/mLeach of three heterodimers (120 μg total protein), 10 mM sodiumphosphate, 150 mM sodium chloride, 10 mM L-Methionine, 5% Sucrose, 0.05%Tween 20 (polysorbate 20) and 0.15% (w/v) aluminium hydroxide at pH6.7±0.2 was determined.

Results

The improved heterodimer, Lip-S4D1-S3hybD1 showed an about 4-fold higheryield in terms of mg/g biomass, a significant improvement overLip-S4D1-S3D1. Additionally, comparable purity of the improvedheterodimer preparation was achievable with one less chromatographystep. (See Table 1.)

TABLE 1 Improved yield of heterodimer Lip S4D1-S3hybD1 compared withLip-S4D1-S3D1. Yield Purity (%) Purity (%) Purity (%) Number ofConstruct (mg/g biomass) RP-HPLC SEC-HPLC SDS-PAGE Chromatography stepsLip-S4D1-S3D1 0.35 88 95 90 5 Lip-S4D1-S3hybD1 2.5 82 96 97 4

Example 3. Immunogenicity of Lipidated Mutant OspA Fragment Heterodimersof Different Serotypes

Immunization of Mice

Female C3H/HeN mice were used for all studies. Prior to immunizations,groups of ten mice were bled via the facial vein and pre-immune serawere prepared and pooled. Three s c immunizations of 100 μL each wereadministered at two week intervals. Each dose contained 1 μg of therespective heterodimer proteins. For the improved heterodimercombination vaccine: Lip-S1D1-S2D1 (SEQ ID NO: 29), Lip-S4D1-S3hybD1(SEQ ID NO: 27) and Lip-S5D1-S6D1 (SEQ ID NO: 33); for the heterodimercombination vaccine of the previous invention: Lip-S1D1-S2D1 (SEQ ID NO:29), Lip-S4D1-S3D1 (SEQ ID NO: 31) and Lip-S5D1-S6D1 (SEQ ID NO: 33) andfor the chimera combination vaccine: Lip-Chimeric OspA ST1/ST2-His (SeqID No: 40), Lip-Chimeric OspA ST5/ST3-His (Seq ID No: 41) andLip-Chimeric OspA ST6/ST4-His (SEQ ID NO: 42). All combination vaccineswere formulated with aluminium hydroxide (Al(OH)₃) at a finalconcentration of 0.15%. One week after the third immunization, blood wascollected from the facial vein and immune sera prepared. In eachexperiment, one group immunized with PBS formulated with Al(OH)₃ wasincluded as a negative control (placebo group). All animal experimentswere conducted in accordance with Austrian law (BGB1 Nr. 501/1989) andapproved by “Magistratsabteilung 58”.

OspA ELISA

ELISA plates (Maxisorp, Nunc, Denmark) were coated with 50 ng (1 μg/mL)protein diluted in coating buffer (PBS) per well and incubated at 4° C.for 16 to 72 hours. The coating antigens were C-terminally His-taggedfull length lipidated OspA ST1-6. The coating buffer was discarded and100 μL blocking buffer (1% BSA, 0.5% Tween-20, PBS) was added andincubated at ambient temperature for 1-2 hours. Plates were washed threetimes with 300 μL (overflow) PBST (0.1% Tween-20, PBS). Five-folddilutions of the sera were made in blocking buffer and 50 μL were addedto each well and incubated for 1 hour at ambient temperature. Plateswere washed three times with 300 μL (overflow) PBST. The secondaryantibody (horseradish peroxidase [HRP]-conjugated rabbit anti-mouse IgG,DAKO, Denmark) was diluted 1:2000 in blocking buffer and 50 μL was addedto each well and incubated for 1 hour at ambient temperature. Plateswere washed three times with 300 μL (overflow) PBST. ABTS(2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), Sigma-Aldrich,USA) was used as substrate for HRP, 50 μL of ABTS was added to each welland incubated for 15 minutes in the dark at ambient temperature. Thereaction was stopped by the addition of 50 μL 1% SDS and the absorbancewas read at 405 nm. A plate was regarded as valid when the absorbance ofthe blank was below 0.1. A sample was valid when the lowest dilution hadan absorbance above 1.0 and the highest dilution was below 0.1. Whenthese criteria were met, the half-max titer was determined. The half-maxtiter is the reciprocal of the dilution that corresponds to the meanabsorbance between the highest and lowest dilutions.

Flow Cytometry

Spirochetes (1×10⁶) were mixed with an equal volume of 4%paraformaldehyde and incubated for 2 hours at room temperature in a96-well plate (Nunclon 96U, Nunc). The plate was centrifuged for 5minutes at 2,000 g and the supernatant was discarded. Cells were washedwith 150 μL HBSS with 2% BSA (HBSS-B), centrifuged as above, and thesupernatant was discarded. Mouse sera were heat inactivated byincubating them at 56° C. for 35 minutes. Heat inactivated sera werediluted in HBSS-B and sterile filtered by centrifuging at 4,000 g for 3minutes using Costar spin-X centrifuge tube filters (0.22 μm, Corning,USA). Spirochetes were dissolved in 100 μL serum and incubated for 45minutes at room temperature. The plate was centrifuged for 15 minutes at2,000 g and the supernatant was discarded. The cells were washed oncewith 150 μL HBSS-B and then resuspended in 100 μL HBSS-B. One microlitersecondary antibody (PE conjugated goat anti-mouse IgG, Beckman Coulter,USA) was added to the cells and incubated at room temperature for 45minutes in the dark. Spirochetes were washed once with 150 μL HBSS-B andthen resuspended in 200 μL HBSS containing 2.5 μM SYTO-17 DNA dye andincubated for 10 minutes at room temperature in the dark. The stainedspirochetes were pelleted by centrifuging for 5 minutes at 2,000 g andsubsequently resuspended in 200 μL HBSS. Labelled spirochetes weremeasured with an FC500 (Beckman Coulter) flow cytometer, gated forSYTO-17 positive events.

Results

Two different OspA heterodimer formulations (“het combo” and “improvedhet combo”) as well as an OspA chimera combination (“chimera combo”)were tested for immunogenicity in mice. Hyperimmune sera were analysedby ELISA for reactivity against full-length OspA (coating antigen) aswell as for surface binding to Borrelia strains expressing differentOspA serotypes (ST1 to ST6).

The ELISA results indicated that all vaccine combinations stimulatedantibody responses to all six OspA serotypes (see FIG. 6 ). It isespecially noteworthy with regard to the current invention that theimproved OspA heterodimer combination vaccine resulted in higher levelsof antibodies specific to serotype 3 OspA in comparison with the OspAheterodimer combination vaccine of the previous invention, whereasantibody levels to other OspA serotypes were comparably stimulated byboth vaccines.

Binding of antibodies from hyperimmune mouse sera directly to borreliaspirochetes was observed in the case of Borreliae expressing all sixOspA serotypes (see FIG. 7 ), indicating that the antibodies generatedin response to all of the antigens are functionally active and can bindnative OspA in situ. The fluorescence intensity was linear over a largerange of serum dilutions. The fluorescence intensity observed inresponse to the improved heterodimer combination vaccine to spirocheteswas comparable to those observed in response to the heterodimercombination vaccine and the chimera combination vaccine. Notably, withregard to binding to serotype 3 OspA borrelia, the antibodies generatedby immunization with the improved vaccine were superior to antibodiesgeneration in response to both of the other combination vaccines.

Example 4. Protective Capacity of the Improved Heterodimer CombinationVaccine Against In Vivo Borrelia Challenge

Immunization of Mice

Female C3H/HeN (H-2^(k)) mice were used for all studies (Janvier,France). Prior to each challenge, groups of five 8-week-old mice werebled via the tail vein and pre-immune sera were prepared and pooled.Three subcutaneous (s c) immunizations of 100 μL were administered attwo week intervals at the doses indicated in Table 2. Both the improvedheterodimer combination vaccine and the chimera combination vaccineincluded three proteins at a ratio of 1:1:1 as described in Example 3.All formulations included aluminium hydroxide (Al(OH)₃) at a finalconcentration of 0.15%. One week after the third immunization, blood wascollected and hyper-immune sera were prepared. In each experiment, onegroup injected with Al(OH)₃ alone (in formulation buffer or PBS) wasincluded as a negative control and one group of mice immunized with thewild-type full-length lipidated OspA protein from the appropriate OspAserotype served as a positive control group (B. burgdorferi strain B31(OspA serotype 1, SEQ ID NO: 34), B. afzelii strain K78 (OspA serotype2, SEQ ID NO: 35), B. garinii strain PHei (OspA serotype 5, SEQ ID NO:38) or B. garinii strain DK29 (OspA serotype 6, SEQ ID NO: 39)). Allanimal experiments were conducted in accordance with Austrian law (BGB1Nr. 501/1989) and approved by “Magistratsabteilung 58”.

Needle Challenge of Immunized Mice with In Vitro Grown Borrelia

Two weeks after the last immunization, mice were challenged s.c. withspirochetes diluted in 100 μL growth medium (BSKII). B. burgdorferistrain ZS7 expressing OspA serotype 1 (experiments 1 and 2), B. gariniistrain PHei expressing OspA serotype 5 (experiments 10 to 13) or B.garinii strain Ma expressing OspA serotype 6 (experiments 14 to 17) wereused for challenge. The challenge doses were strain-dependent anddependent on the virulence of the individual strains, which was assessedby challenge experiments for determination of ID₅₀. Doses employed forneedle challenge experiments ranged from 20 to 50 times the ID₅₀. Priorto each challenge, OspA expression was verified by flow cytometry (seeexample 3). Challenge of mice was only performed with cultureswhere >80% of cells were positive for OspA expression.

Challenge of Immunized Mice with Ticks Infected with B. burgdorferi orB. afzelii (“Tick Challenge”)

Two weeks after the last immunization, mice were challenged with ticksharboring B. burgdorferi strain Pra4 expressing OspA serotype 1(experiment 3), B. burgdorferi strain Pra1 expressing OspA serotype 1(experiments 4 and 5) or B. afzelii expressing OspA serotype 2(experiments 6 to 9). In order to facilitate tick infection of theimmunized mice, the hair of the back of each mouse was removed withVeet® Cream (Reckitt Benckiser, United Kingdom) and a small ventilatedcontainer was glued to the skin with super glue (Pattex, Germany)Thereafter, two to three I. ricinus nymphs infected with B. burgdorferistrain Pra1 or Pra 4 or B. afzelii strain IS1 were applied per mouse andallowed to attach and feed until they were fully engorged and droppedoff. The feeding status was monitored daily for each individual tick.Only those mice from which at least one fully- or almost fully-fed tickwas collected were included in the final readout.

Sacrifice of Mice and Collection of Material

Four or six weeks after needle or tick challenge, respectively, micewere sacrificed by cervical dislocation. Blood was collected by orbitalbleeding and final sera were prepared and used for VlsE ELISA and/orwestern blot to determine infection status. In addition, the urinarybladder from each mouse was collected and DNA was extracted andsubjected to quantitative PCR (qPCR) for identification of Borrelia.

ELISA with the Invariable Region 6 (IR6) of VlsE

A biotinylated 25-mer peptide (MKKDDQIAAAMVLRGMAKDGQFALK, SEQ ID NO: 59)derived from the sequence of B. garinii strain IP90 was used for theanalysis (Liang F T, Alvarez A L, Gu Y, Nowling J M, Ramamoorthy R,Philipp M T. An immunodominant conserved region within the variabledomain of VlsE, the variable surface antigen of Borrelia burgdorferi. JImmunol. 1999; 163:5566-73). Streptavidin pre-coated 96-well ELISAplates (Nunc), were coated with 100 μL/well (1 μg/mL) peptide in PBSsupplemented with 0.1% Tween (PBS/0.1 T). The plates were incubatedovernight at 4° C. After coating with the peptide, the plates werewashed once with PBS/0.1 T. The plates were then blocked for one hour atroom temperature (RT) with 100 μL/well of PBS+2% BSA, before beingwashed again with PBS/0.1 T. Reactivity of post-challenge sera (finalsera) to the peptide was tested at 1:200 and 1:400 dilutions in PBS+1%BSA. Plates were incubated for 90 minutes at RT before being washedthree times with PBS/0.1 T. Each well then received 50 μL of 1.3 μg/mLpolyclonal rabbit anti-mouse IgG conjugated to HRP (Dako) in PBS+1% BSA.The plates were then incubated for 1 hour at RT. After three washes withPBS/0.1 T, ABTS (50 μL/well) was added as substrate (Sigma-Aldrich) andcolor was allowed to develop for 30 minutes. Absorbance was measured at405 nm. All sera were tested in duplicate. Negative controls includedPBS instead of sera as well as plates not coated with the peptide. Serafrom mice shown to be culture positive for borrelia infection were usedas positive controls.

DNA Extraction and Purification

The urinary bladder from each mouse was subjected to DNA extraction andpurification using the DNeasy Blood and Tissue Kit (Qiagen) according tothe manufacturer's instructions with the following modification. Eachurinary bladder was digested overnight at 60° C. using 100 μLrecombinant Proteinase K (PCR grade; 14-22 mg/mL, Roche). The DNA waseluted in 50 μL sterile deionized water and stored at −20° C. As anegative control, every tenth sample was followed by one emptypurification column in each DNA extraction and purification.

qPCR Targeting recA

Oligonucleotide primers were designed for the recA gene in a manner thatthey could be used in qPCR for identification of all relevant Borreliaspecies causing Lyme borreliosis (forward: CATGCTCTTGATCCTGTTTA, SEQ IDNO: 57, reverse: CCCATTTCTCCATCTATCTC, SEQ ID NO: 58). The recA fragmentwas cloned from the B. burgdorferi s.s. strain N40 into pET28b(+), to beused as standard in each reaction. The chromosomal DNA extracted frommouse urinary bladders was diluted 1:4 in water in order to reducematrix effects observed with undiluted DNA. A master mix consisting of10 μL SSoAdvanced™ SYBR® Green Supermix, 0.3 μL of each primer (10 μM),and 7.4 μL water was prepared for each experiment. Eighteen μL of mastermix was mixed with 2 μL of the diluted DNA extracted from urinarybladder in micro-titer plates and the DNA was amplified using a CFX96real-time PCR detection system (Bio-Rad). The DNA was denatured for 3minutes at 95° C., followed by 50 cycles of 15 seconds at 95° C. and 30seconds at 55° C. After amplification, the DNA was prepared for meltingcurve analysis by denaturation for 30 seconds at 95° C. followed by 2minutes at 55° C. The melting curve analysis was performed by 5 secondsincubation at 55° C., with a 0.5° C. increase per cycle, and 5 secondsat 95° C. On each plate, four no-template controls (NTC) were includedas well as a standard curve in duplicate with template copy numbersranging from 10 to 10,000.

Western Blot

Binding of final sera to whole cell lysates from borrelia belonging tothe corresponding OspA serotype was analyzed by western blot. Briefly,2.5 μg of spirochete lysate per mouse sera to be analyzed was separatedby SDS-PAGE under reducing conditions using 4-12% Tris-Glycine ZOOM gels(Invitrogen). Separated proteins were transferred onto a nitrocellulosemembrane using the iBlot® Dry blotting system (Invitrogen). Afterblocking in 5% milk for 1 hour, final sera were added at a 1:2000dilution and incubated at +4° C. overnight. The membranes were thenwashed three times with PBS/0.1 T followed by a one hour incubation inpolyclonal rabbit anti mouse IgG conjugated to HRP (Dako) diluted1:10,000. The immunoblots were visualized with Amersham ECL Plus'Western blotting detection reagents (GE Healthcare) and Kodak BioMaxfilms (Kodak).

Infection Readout

The final infection readout was based on two separate methods: detectingthe presence of Borrelia-specific antibodies (western blot and VlsEELISA) and presence of Borrelia DNA (qPCR targeting recA). Inexperiments where B. burgdorferi strain ZS7 was used for challenge(experiments 1 and 2), western blot together with qPCR were applied. Inall other experiments, VlsE ELISA and qPCR were used. There was a highconsistency between the two methods (>95%); therefore a mouse wasregarded as infected when at least one of the two methods was positive.Statistical significance was determined by Fisher's exact test(two-tailed).

Results

The improved heterodimer combination vaccine was tested for protectivecapacity against Borrelia challenge. The results of these experimentsare summarized in Table 2 Immunized mice were challenged with B.burgdorferi s.s. (OspA serotype 1, strain ZS7, needle challenge,Experiments 1 and 2 or strains Pra1 or Pra 4, tick challenge,Experiments 3 or 4 and 5, respectively), B. afzelii (OspA serotype 2,strain IS1, tick challenge, Experiments 6-9), B. garinii (OspA serotype5, strain PHei, needle challenge, Experiments 10-13) or B. garinii (OspAserotype 6, strain Ma, needle challenge, Experiments 14-17). In someexperiments, other OspA-based antigens, such as the chimera combinationvaccine or a lipidated full-length OspA protein, were included. A groupof mice immunized with PBS or formulation buffer combined with Al(OH)₃served as a placebo (adjuvant alone) control group in each experiment.

The protection data from the 17 experiments are summarized in Table 2.In all experiments, a high level of infection was seen in all placebogroups. Additionally, a low infection rate was observed in the groupsreceiving the corresponding full length OspA protein, with the exceptionof the full-length OspA serotype 6, wherein only partial protection wasobserved (experiments 14 to 17). These results validate the experimentalset-up and readout methods.

The improved heterodimer combination vaccine conferred significantprotection (p-values<0.05), at a 3 μg dose, when mice were challengedwith in vitro grown B. burgdorferi s.s. or B. garinii (OspA serotype 5or 6), or ticks harboring B. burgdorferi or B. afzelii. Furthermore,when different immunization doses were assessed for vaccine efficacy,highly significant protection (p-values<0.01) could be shown when 0.03μg of the improved heterodimer combination vaccine was administered andthe mice were challenged with B. afzelii or B. garinii (OspA serotype 5or 6) (Experiment 8, 9, 12, 13 and 16). In summary, the improvedheterodimer combination vaccine induced protective immunity againstthree Borrelia species (B. burgdorferi, B. afzelii and B. garinii)including four clinically relevant OspA serotypes (1, 2, 5 and 6), asshown in mouse models using either in vitro grown spirochetes orinfected ticks for challenge.

Protection against serotypes 5 and 6 comparable to that conferred by theimproved heterodimer combination vaccine was also observed in miceimmunized with the chimera combination vaccine (data not shown).

TABLE 2 Protective capacity of the improved mutant OspA heterodimercombination vaccine of the invention against OspA serotype 1, serotype2, serotype 5 and serotype 6 Borrelia challenge. Groups of mice wereimmunized three times with the indicated doses of immunogen or Al(OH)₃adjuvant alone at two-week intervals. Immunogens used were a 1:1:1combination of the mutant OspA heterodimers Lip-S1D1-S2D1,Lip-S4D1-S3hybD1 and Lip-S5D1-S6D1 (“Improved heterodimer combinationvaccine”), a 1:1:1 combination of Lip-Chimeric OspA ST1/ST2-His,Lip-Chimeric OspA ST5/ST3-His and Lip-Chimeric OspA ST6/ST4-His(“Chimera combination vaccine”) and Lip-OspA1-His (lipidated full-lengthOspA protein from B. burgdorferi strain B31) or Lip-OspA2-His (lipidatedfull-length OspA protein from B. afzelii strain K78), Lip-OspA5-His(lipidated full-length OspA protein from B. garinii strain PHei) orLip-OspA6-His (lipidated full-length OspA protein from B. garinii strainDK29). Immunized mice were challenged s.c. two weeks after the lastimmunization with the indicated borrelia species using a syringe (B.burgdorferi strain ZS7, B. garinii strain PHei or B. garinii strain Ma)or using ticks (B. burgdorferi strain Pra1 or Pra4 or B. afzelii strainIS1). A Protection against needle challenge with serotype 1 OspAborrelia by chimera combination vaccine and improved heterodimercombination vaccine (one dose: 3 μg) Experiment 1: Experiment 2:Infected/Total Infected/Total Immunogen Dose Challenge (p-value)(p-value) Lip-OspA1-His (SEQ ID NO: 34) 3 × 1.0 μg B. burgdorferi 0/10(<0.0001) 1/10 (<0.0001) (OspA serotype 1) strain ZS7 Chimeracombination vaccine: Lip-Chimeric OspA ST1/ST2-His 3 × 1.0 μg B.burgdorferi 0/10 (<0.0001) 0/10 (<0.0001) (Seq ID No: 40) (OspAserotype 1) Lip-Chimeric OspA ST5/ST3-His 3 × 1.0 μg strain ZS7 (Seq IDNo: 41) Lip-Chimeric OspA ST6/ST4-His 3 × 1.0 μg (Seq ID No: 42)Improved heterodimer combination vaccine: Lip-S1D1-S2D1 (Seq ID No: 29)3 × 1.0 μg B. burgdorferi 0/10 (<0.0001) 0/10 (<0.0001) Lip-S4D1-S3hybD1(Seq ID No: 27) 3 × 1.0 μg (OspA serotype 1) Lip-S5D1-S6D1 (Seq ID No:33) 3 × 1.0 μg strain ZS7 Al(OH)₃ adjuvant alone — B. burgdorferi 9/910/10 (OspA serotype 1) strain ZS7 B Protection against tick challengewith serotype 1 OspA borrelia by chimera combination vaccine andimproved heterodimer combination vaccine (one dose: 3 μg) Experiment 3:Experiment 4: Infected/Total Infected/Total Immunogen Dose Challenge(p-value) (p-value) Lip-OspA1-His (SEQ ID NO: 34) 3 × 1.0 μg Tickchallenge with 0/4 (0.0475) 1/9 (0.0216) B. burgdorferi (OspAserotype 1) strain Pra4 (Exp. 3) or Pra1 (Exp. 4) Chimera combinationvaccine: Lip-Chimeric OspA ST1/ST2-His 3 × 1.0 μg Tick challenge with0/8 (0.0060) 0/7 (0.0093) (Seq ID No: 40) B. burgdorferi Lip-ChimericOspA ST5/ST3-His 3 × 1.0 μg (OspA serotype 1) (Seq ID No: 41) strainPra4 (Exp. 3) Lip-Chimeric OspA ST6/ST4-His 3 × 1.0 μg or Pra1 (Exp. 4)(Seq ID No: 42) Improved heterodimer combination vaccine: Lip-S1D1-S2D1(Seq ID No: 29) 3 × 1.0 μg Tick challenge with 0/5 (0.0260) 0/7 (0.0093)Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 × 1.0 μg B. burgdorferi Lip-S5D1-S6D1(Seq ID No: 33) 3 × 1.0 μg (OspA serotype 1) strain Pra4 (Exp. 3) orPra1 (Exp. 4) Al(OH)₃ adjuvant alone — Tick challenge with 5/6 5/6 B.burgdorferi (OspA serotype 1) strain Pra4 (Exp. 3) or Pra1 (Exp. 4) CProtection against tick challenge with serotype 1 OspA borrelia byimproved heterodimer combination vaccine (decreasing doses: 3 μg and 0.3μg) Experiment 5: Infected/Total Immunogen Dose Challenge (p-value)Improved heterodimer combination vaccine: Lip-S1D1-S2D1 (Seq ID No: 29)3 × 1.0 μg Tick challenge with 1/7 (0.0174) Lip-S4D1-S3hybD1 (Seq ID No:27) 3 × 1.0 μg B. burgdorferi Lip-S5D1-S6D1 (Seq ID No: 33) 3 × 1.0 μg(OspA serotype 1) strain Pra1 Improved heterodimer combination vaccine:Lip-S1D1-S2D1 (Seq ID No: 29) 3 × 0.1 μg Tick challenge with 1/9(0.0059) Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 × 0.1 μg B. burgdorferiLip-S5D1-S6D1 (Seq ID No: 33) 3 × 0.1 μg (OspA serotype 1) strain Pra1Al(OH)₃ adjuvant alone — Tick challenge with 7/8 B. burgdorferi (OspAserotype 1) strain Pra1 D Protection against tick challenge withserotype 2 OspA borrelia by chimera combination vaccine and improvedheterodimer combination vaccine (one dose: 3 μg) Experiment 6:Experiment 7: Infected/Total Infected/Total Immunogen Dose Challenge(p-value) (p-value) Lip-OspA2-His (SEQ ID NO: 35) 3 × 1.0 μg Tickchallenge with 0/8 (0.0016) 0/9 (0.0002) B. afzelii (OspA serotype 2)strain IS1 Chimera combination vaccine: Lip-Chimeric OspA ST1/ST2-His 3× 1.0 μg Tick challenge with 0/7 (0.0025) 0/9 (0.0002) (Seq ID No: 40)B. afzelii (OspA Lip-Chimeric OspA ST5/ST3-His 3 × 1.0 μg serotype 2)strain (Seq ID No: 41) IS1 Lip-Chimeric OspA ST6/ST4-His 3 × 1.0 μg (SeqID No: 42) Improved heterodimer combination vaccine: Lip-S1D1-S2D1 (SeqID No: 29) 3 × 1.0 μg Tick challenge with 0/9 (0.0010) 0/7 (0.0003)Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 × 1.0 μg B. afzelii (OspALip-S5D1-S6D1 (Seq ID No: 33) 3 × 1.0 μg serotype 2) strain IS1 Al(OH)₃adjuvant alone — Tick challenge with 5/5 8/8 B. afzelii (OspA serotype2) strain IS1 E Protection against tick challenge with serotype 2 OspAborrelia by improved heterodimer combination vaccine (decreasing doses:0.03 μg and 0.003 μg) Experiment 8: Experiment 9: Infected/TotalInfected/Total Immunogen Dose Challenge (p-value) (p-value) Improvedheterodimer combination vaccine: Lip-S1D1-S2D1 (Seq ID No: 29) 3 × 0.01μg Tick challenge with 0/9 (0.0004) 2/7 (0.0096) Lip-S4D1-S3hybD1 (SeqID No: 27) 3 × 0.01 μg B. afzelii (OspA Lip-S5D1-S6D1 (Seq ID No: 33) 3× 0.01 μg serotype 2) strain IS1 Improved heterodimer combinationvaccine: Lip-S1D1-S2D1 (Seq ID No: 29) 3 × 0.001 μg Tick challenge with7/10 (n.s.) 1/8 (0.0008) Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 × 0.001 μgB. afzelii (OspA Lip-S5D1-S6D1 (Seq ID No: 33) 3 × 0.001 μg serotype 2)strain IS1 Al(OH)₃ adjuvant alone — Tick challenge with 6/6 9/9 B.afzelii (OspA serotype 2) strain IS1 F Protection against needlechallenge with serotype 5 OspA borrelia by improved heterodimercombination vaccine (one dose: 3 μg) Experiment 10: Experiment 11:Infected/Total Infected/Total Immunogen Dose Challenge (p-value)(p-value) Lip-OspA5-His (SEQ ID NO: 38) 3 × 1.0 μg B. garinii (OspA 0/10(<0.0001) 0/10 (<0.0001) serotype 5) strain PHei) Improved heterodimercombination vaccine: Lip-S1D1-S2D1 (Seq ID No: 29) 3 × 1.0 μg B. garinii(OspA 0/10 (<0.0001) 1/10 (<0.0001) Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 ×1.0 μg serotype 5) strain Lip-S5D1-S6D1 (Seq ID No: 33) 3 × 1.0 μg PHei)Al(OH)₃ adjuvant alone — B. garinii (OspA 10/10 10/10 serotype 5) strainPHei) G Protection against needle challenge with serotype 5 OspAborrelia by improved heterodimer combination vaccine (decreasing doses:3 μg, 0.3 μg and 0.03 μg) Experiment 12: Experiment 13: Infected/TotalInfected/Total Immunogen Dose Challenge (p-value) (p-value)Lip-OspA5-His (SEQ ID NO: 38) 3 × 1.0 μg B. garinii (OspA 0/10 (0.0007)  0/10 (<0.001) serotype 5) strain PHei) Improved heterodimer combinationvaccine: Lip-S1D1-S2D1 (Seq ID No: 29) 3 × 1.0 μg B. garinii (OspA 0/10(<0.0007) 1/10 (0.0002) Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 × 1.0 μgserotype 5) strain Lip-S5D1-S6D1 (Seq ID No: 33) 3 × 1.0 μg PHei)Improved heterodimer combination vaccine: Lip-S1D1-S2D1 (Seq ID No: 29)3 × 0.1 μg B. garinii (OspA 0/10 (<0.0007)  0/10 (<0.0001)Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 × 0.1 μg serotype 5) strainLip-S5D1-S6D1 (Seq ID No: 33) 3 × 0.1 μg PHei) Improved heterodimercombination vaccine: Lip-S1D1-S2D1 (Seq ID No: 29) 3 × 0.01 μg B.garinii (OspA 2/10 (<0.0219) 3/10 (0.0047) Lip-S4D1-S3hybD1 (Seq ID No:27) 3 × 0.01 μg serotype 5) strain Lip-S5D1-S6D1 (Seq ID No: 33) 3 ×0.01 μg PHei) Al(OH)₃ adjuvant alone — B. garinii (OspA 8/10 9/9serotype 5) strain PHei) H Protection against needle challenge withserotype 6 OspA borrelia by improved heterodimer combination vaccine(one dose: 3 μg) Experiment 14: Experiment 15: Infected/TotalInfected/Total Immunogen Dose Challenge (p -value) (p-value)Lip-OspA6-His (SEQ ID NO: 39) 3 × 1.0 μg B. garinii (OspA 4/10 (0.0108) 6/10 (n.s.) serotype 6) strain Ma) Improved heterodimer combinationvaccine: Lip-S1D1-S2D1 (Seq ID No: 29) 3 × 1.0 μg B. garinii (OspA 0/10(<0.0001) 0/10 (0.0007) Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 × 1.0 μgserotype 6) strain Lip-S5D1-S6D1 (Seq ID No: 33) 3 × 1.0 μg Ma) Al(OH)₃adjuvant alone — B. garinii (OspA 10/10 8/10 serotype 6) strain Ma) IProtection against needle challenge with serotype 6 OspA borrelia byimproved heterodimer combination vaccine (decreasing doses: 3 μg, 0.3 μgand 0.03 μg) Experiment 16: Experiment 17: Infected/Total Infected/TotalImmunogen Dose Challenge (p-value) (p-value) Lip-OspA6-His (SEQ ID NO:39) 3 × 1.0 μg B. garinii (OspA 4/10 (0.0108) 8/10 (n.s.) serotype 6)strain Ma) Improved heterodimer combination vaccine: Lip-S1D1-S2D1 (SeqID No: 29) 3 × 1.0 μg B. garinii (OspA 0/10 (<0.0001) 0/10 (0.0001)Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 × 1.0 μg serotype 6) strainLip-S5D1-S6D1 (Seq ID No: 33) 3 × 1.0 μg Ma) Improved heterodimercombination vaccine: Lip-S1D1-S2D1 (Seq ID No: 29) 3 × 0.1 μg B. garinii(OspA 2/10 (0.0007) 2/10 (0.0054) Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 ×0.1 μg serotype 6) strain Lip-S5D1-S6D1 (Seq ID No: 33) 3 × 0.1 μg Ma)Improved heterodimer combination vaccine: Lip-S1D1-S2D1 (Seq ID No: 29)3 × 0.01 μg B. garinii (OspA 3/10 (0.0031) 4/10 (n.s.) Lip-S4D1-S3hybD1(Seq ID No: 27) 3 × 0.01 μg serotype 6) strain Lip-S5D1-S6D1 (Seq ID No:33) 3 × 0.01 μg Ma) Al(OH)₃ adjuvant alone — B. garinii (OspA 10/10 9/10serotype 6) strain Ma) P-value; Fisher's exact test, two-tailed, ascompared to the adjuvant alone group, are indicated in parenthesis. notsignificant (n.s.).

SEQUENCES S3hybD1: hybrid OspA C-terminal fragment; amino acids ofpositions 125-176 from Borrelia valaisiana, strain VS116,and amino acids 177-274 from Borrelia garinii, strain PBr,with disulfide bond type 1 and T in position 233 SEQ ID NO: 1FNEKGEVSEKILTRSNGTTLEYSQMTDAENATKAVETLKNGIKLPGNLVGGKTKLTVT C GTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQK T KQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAEL C AALKB. valaisiana (strain VS116), OspA aa 125-176 SEQ ID NO: 2FNEKGEVSEKILTRSNGTTLEYSQMTDAENATKAVETLKNGIKLPGNLVGGKB. garinii (strain PBr, serotype 3), OspA aa 177-274, with T inposition 233, from full-length OspA (SEQ ID NO: 8) SEQ ID NO: 3TKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQK T KQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALK B. valaisiana (strain VS116), OspASEQ ID NO: 4MKKYLLGIGLILALIACKQNVSSLDEKNSASVDLPGEMKVLVSKEKDKDGKYSLVATVDKVELKGTSDKNNGSGTLEGVKDDKSKVKLTISDDLGETKLETFKEDGTLVSRKVNFKDKSFTEEKFNEKGEVSEKILTRSNGTTLEYSQMTDAENATKAVETLKNGIKLPGNLVGGKTTLKITEGTVTLSKHIAKSGEVTVEINDTSSTPNTKKTGKWDARNSTLTIIVDSKNKTKLVFTKQDTITVQSYNPAGNKLEGTAVEIKTLQELKNALKB. burgdorferi s.s. (strain B31, OspA serotype 1) SEQ ID NO: 5MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDGKYDLIATVDKLELKGTSDKNNGSGVLEGVKADKSKVKLTISDDLGQTTLEVFKEDGKTLVSKKVTSKDKSSTEEKFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKGYVLEGTLTAEKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEIKNALK B. afzelii (strain K78; OspA serotype 2)SEQ ID NO: 6MKKYLLGIGLILALIACKQNVSSLDEKNSASVDLPGEMKVLVSKEKDKDGKYSLKATVDKIELKGTSDKDNGSGVLEGTKDDKSKAKLTIADDLSKTTFELFKEDGKTLVSRKVSSKDKTSTDEMFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALKB. garinii (strain PBr, OspA serotype 3) with P in position 233(embl accession X80256.1) SEQ ID NO: 7MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGGMKVLVSKEKDKDGKYSLMATVEKLELKGTSDKSNGSGVLEGEKADKSKAKLTISQDLNQTTFEIFKEDGKTLVSRKVNSKDKSSTEEKFNDKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQK P KQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELKAALKB. garinii (strain PBr, OspA serotype 3) with T in position 233(embl accession ACL34827.1) SEQ ID NO: 8MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGGMKVLVSKEKDKDGKYSLMATVEKLELKGTSDKSNGSGVLEGEKADKSKAKLTISQDLNQTTFEIFKEDGKTLVSRKVNSKDKSSTEEKFNDKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQK T KQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELKAALK B. bavariensis (strain PBi, OspA serotype 4)SEQ ID NO: 9MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGEMKVLVSKEKDKDGKYSLMATVDKLELKGTSDKSNGSGTLEGEKSDKSKAKLTISEDLSKTTFEIFKEDGKTLVSKKVNSKDKSSIEEKFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELKNALK B. garinii (strain PHei, OspA serotype 5)SEQ ID NO: 10MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGGMKVLVSKEKDKDGKYSLMATVEKLELKGTSDKNNGSGTLEGEKTDKSKVKLTIAEDLSKTTFEIFKEDGKTLVSKKVTLKDKSSTEEKFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELKNALK B. garinii (strain DK29, OspA serotype 6)SEQ ID NO: 11MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGGMTVLVSKEKDKDGKYSLEATVDKLELKGTSDKNNGSGTLEGEKTDKSKVKSTIADDLSQTKFEIFKEDGKTLVSKKVTLKDKSSTEEKFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELKNALK B. garinii (strain T25, OspA serotype 7)SEQ ID NO: 12MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGEMKVLVSKEKDKDGKYSLEATVDKLELKGTSDKNNGSGVLEGVKAAKSKAKLTIADDLSQTKFEIFKEDGKTLVSKKVTLKDKSSTEEKFNDKGKLSEKVVTRANGTRLEYTEIQNDGSGKAKEVLKSLTLEGTLTADGETKLTVEAGTVTLSKNISESGEITVELKDTETTPADKKSGTWDSKTSTLTISKNSQKTKQLVFTKENTITVQKYNTAGTKLEGSPAEIKDLEALKAALK Borrelia OspA lipidation signal SEQ ID NO: 13MKKYLLGIGLILALIA Borrelia OspB lipidation signal SEQ ID NO: 14MRLLIGFALALALIG E. coli lpp lipidation signal SEQ ID NO: 15MKATKLVLGAVILGSTLLAGLN1 peptide linker constructed from two separate loop regionsof the N-terminal half of OspA from B. burgdorferi s.s. strainB31 (aa 65-74 and aa 42-53, amino acid exchange at position 53: D535)SEQ ID NO: 16 GTSDKNNGSGSKEKNKDGKYShLFA-1-like sequence from B. burgdorferi s.s. strain B31(OspA serotype 1) SEQ ID NO: 17 GYVLEGTLTAENon-hLFA-1-like sequence from B. afzelii strain K78 (OspA serotype 2)SEQ ID NO: 18 NFTLEGKVANDB. burgdorferi s.s. (strain B31, serotype 1), OspA aa 126-273with replaced hLFA-like sequence from serotype 1 OspA SEQ ID NO: 19FNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEIKNALKB. afzelii (strain K78, serotype 2), OspA aa 126-273 SEQ ID NO: 20FNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALKB. garinii (strain PBr, serotype 3), OspA aa 126-274 SEQ ID NO: 21FNDKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELKAALKB. bavariensis (strain PBi, serotype 4), OspA aa 126-273 SEQ ID NO: 22FNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELKNALKB. garinii (strain PHei, serotype 5), OspA aa 126-273 SEQ ID NO: 23FNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELKNALKB. garinii (strain DK29, serotype 6), OspA aa 126-274 SEQ ID NO: 24FNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELKNALKB. garinii (strain T25, serotype 7) OspA aa 126-274 SEQ ID NO: 25FNDKGKLSEKVVTRANGTRLEYTEIQNDGSGKAKEVLKSLTLEGTLTADGETKLTVEAGTVTLSKNISESGEITVELKDTETTPADKKSGTWDSKTSTLTISKNSQKTKQLVFTKENTITVQKYNTAGTKLEGSPAEIKDLEALKAALKLip-S4D1-S3hybD1-nt Coding sequence for intermediate and finalheterodimer fusion proteins of OspA serotype 4 and OspAserotype 3 with disulfide bond type 1, E. coli lpp lipidationsignal, LN1 linker sequence, serotype 3 OspA fragment comprisingamino acids 125-176 of B. valaisiana, strain VS116 (SEQ ID NO: 2)and amino acids 177-274 of B. garinii, strain PBr, serotype3 (SEQ ID NO: 3) SEQ ID NO: 26ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAATGCTAAGGGCGAACTGAGCGAAAAAACGATCCTGCGTGCGAATGGCACCCGTCTGGAATACACCGAAATCAAATCCGATGGTACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCAACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAAGATACGATCACCGTTCAAAAATATGACTCCGCGGGCACCAACCTGGAAGGCAATGCCGTCGAAATCAAAACCCTGGATGAACTGTGTAACGCCCTGAAGGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAGTGAGCGAAAAAATTCTGACCCGTAGCAATGGCACCACCCTGGAATATAGCCAGATGACCGATGCAGAAAATGCAACCAAAGCAGTTGAAACCCTGAAAAACGGTATTAAACTGCCTGGTAATCTGGTTGGTGGTAAAACCAAACTGACCGTTACCTGTGGCACCGTTACCCTGAGCAAAAACATTAGCAAAAGCGGTGAAATTACCGTGGCACTGAATGATACCGAAACCACACCGGCAGACAAAAAAACCGGTGAATGGAAAAGCGATACCAGCACCCTGACCATTAGTAAAAATAGCCAGAAAACAAAACAGCTGGTGTTTACCAAAGAAAACACCATTACCGTGCAGAATTATAACCGTGCAGGTAATGCACTGGAAGGTAGTCCGGCAGAAATTAAAGATCTGGCAGAACTGTGTGCAGCCCTGAAATAALip-S4D1-S3hybD1-aa: Heterodimer fusion protein of OspAserotype 4 and OspA serotype 3, comprising amino acids125-176 of B. valaisiana, strain VS116 (SEQ ID NO: 2) andamino acids 177-274 of B. garinii, strain PBr, serotype 3(SEQ ID NO: 3), with disulfide bond type 1, N-terminalCSS for addition of lipids, LN1 linker sequence, N-terminal lipidationSEQ ID NO: 27LipCSSFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEVSEKILTRSNGTTLEYSQMTDAENATKAVETLKNGIKLPGNLVGGKTKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKTKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALK Lip-S1D1-S2D1-nt: Coding sequence for intermediate and finalheterodimer fusion proteins of OspA serotype 1 and OspAserotype 2 with disulfide bond type 1, E. coli lpp lipidationsignal, LN1 linker sequence, aa 164-174 of OspA serotype 1replaced by non-hLFA-1-like sequence NFTLEGKVAND SEQ ID NO: 28ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAGTCAGCGAAAAAATCATTACCCGCGCAGACGGCACCCGCCTGGAATACACCGGCATCAAATCGGACGGCAGCGGCAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAAACCACCCTGGTGGTGAAATGCGGCACCGTTACGCTGAGCAAAAACATTAGTAAATCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAGAAAACCGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATAGCAAGAAAACCAAAGATCTGGTCTTCACGAAAGAAAACACCATCACGGTGCAGCAATATGACAGCAATGGTACCAAACTGGAAGGCTCCGCTGTGGAAATCACGAAACTGGATGAAATCTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAAAACGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAATGCGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAATAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAAGATACGATCACCGTGCAGAAATACGACAGTGCGGGTACCAACCTGGAAGGCACGGCTGTTGAAATCAAAACCCTGGACGAACTGTGTAACGCCCTGAAALip-S1D1-S2D1-aa: Heterodimer fusion protein of OspA serotype1 and OspA serotype 2 with disulfide bond type 1, N-terminalCSS for addition of lipids, LN1 linker sequence, aa 164-174 ofOspA serotype 1 replaced by non-hLFA-1-like sequenceNFTLEGKVAND, N-terminal lipidation SEQ ID NO: 29LipCSSFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEICNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTT QATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALKLip-S4D1-S3D1-nt: Coding sequence for intermediate and finalheterodimer fusion proteins of OspA serotypes 4 and 3 bothwith disulfide bond type 1, E. coli lpp lipidation signal,N-terminal CSS for addition of lipids, LN1 linker sequence SEQ ID NO: 30ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAATGCTAAGGGCGAACTGAGCGAAAAAACGATCCTGCGTGCGAATGGCACCCGTCTGGAATACACCGAAATCAAATCCGATGGTACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCAACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAAGATACGATCACCGTTCAAAAATATGACTCCGCGGGCACCAACCTGGAAGGCAATGCCGTCGAAATCAAAACCCTGGATGAACTGTGTAACGCCCTGAAGGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTTAACGATAAGGGCAAACTGTCGGAAAAAGTGGTCACCCGCGCAAATGGCACCCGCCTGGAATACACGGAAATCAAAAACGATGGTAGCGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGTGCGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAAAACACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCACCGGCTGAAATCAAGGACCTGGCTGAACTGTGTGCGGCACTGAAA Lip-S4D1-S3D1-aa: Heterodimer fusion protein of OspAserotypes 4 and 3 both with disulfide bond type 1, N-terminal CSS for addition of lipids, LN1 linker sequence,N-terminal lipidation SEQ ID NO: 31LipCSSFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNDKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALK Lip-S5D1-S6D1-nt: Coding sequence for intermediate and finalheterodimer fusion proteins of OspA serotypes 6 both withdisulfide bond type 1, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 linker sequence SEQ ID NO: 32ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAATCTCAGAAAAAACCATCGTCCGCGCTAACGGCACCCGCCTGGAATACACCGACATCAAATCAGACAAGACCGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCTGCGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGATTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAAGATACGATCACCGTGCAAAACTATGACAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGGCAAAGGTGAAACGAGCGAAAAGACCATCGTGCGTGCGAACGGTACCCGCCTGGAATATACGGACATTAAATCGGACGGCAGCGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGATAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAAGATACGATCACCGTTCAACGCTATGACAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAA Lip-55D1-56D1-aa: Heterodimer fusion protein of OspAserotypes 6 both with disulfide bond type 1, N-terminalCSS for addition of lipids, LN1 linker sequence, N-terminal lipidationSEQ ID NO: 33LipCSSFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALK B. burgdorferi (strain B31, OspA serotype 1) aa 18-273,lpp lipidation signal sequence removed (MKATKLVLGAVILGSTLLAG,SEQ ID NO: 15), C-terminal His tag (LEHHHHHH), N-terminalCSSF for addition of lipids SEQ ID NO: 34CSSFKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDGKYDLIATVDKLELKGTSDKNNGSGVLEGVKADKSKVKLTISDDLGQTTLEVFKEDGKTLVSKKVTSKDKSSTEEKFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKGYVLEGTLTAEKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEIKNALKLEHHHHHHB. afzelii (strain K78; OspA serotype 2) aa 18-273, lpplipidation signal sequence removed (MKATKLVLGAVILGSTLLAG,SEQ ID NO: 15), C-terminal His tag (LEHHHHHH), N-terminalCSSF for addition of lipids SEQ ID NO: 35CSSFKQNVSSLDEKNSASVDLPGEMKVLVSKEKDKDGKYSLKATVDKIELKGTSDKDNGSGVLEGTKDDKSKAKLTIADDLSKTTFELFKEDGKTLVSRKVSSKDKTSTDEMFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALKLEHHHHHHB. garinii (strain PBr; OspA serotype 3) aa 18-274, lpplipidation signal sequence removed (MKATKLVLGAVILGSTLLAG,SEQ ID NO: 15), C-terminal His tag (LEHHHHHH), N-terminalCSSF for addition of lipids SEQ ID NO: 36CSSFKQNVSSLDEKNSVSVDLPGGMKVLVSKEKDKDGKYSLMATVEKLELKGTSDKSNGSGVLEGEKADKSKAKLTISQDLNQTTFEIFKEDGKTLVSRKVNSKDKSSTEEKFNDKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKTKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELKAALKLEHHHHHHB. bavariensis (strain PBi; OspA serotype 4) aa 18-273, lpplipidation signal sequence removed (MKATKLVLGAVILGSTLLAG, SEQID NO: 15), C-terminal His tag (LEHHHHHH), N-terminalCSSF for addition of lipids SEQ ID NO: 37CSSFKQNVSSLDEKNSVSVDLPGEMKVLVSKEKDKDGKYSLMATVDKLELKGTSDKSNGSGTLEGEKSDKSKAKLTISEDLSKTTFEIFKEDGKTLVSKKVNSKDKSSIEEKFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELKNALKLEHHHHHHB. garinii (strain PHei; OspA serotype 5) aa 18-273, lpplipidation signal sequence removed (MKATKLVLGAVILGSTLLAG,SEQ ID NO: 15), C-terminal His tag (LEHHHHHH, SEQ ID NO:64), N-terminal CSSF (SEQ ID NO: 65) for addition of lipidsSEQ ID NO: 38CSSFKQNVSSLDEKNSVSVDLPGGMKVLVSKEKDKDGKYSLMATVEKLELKGTSDKNNGSGTLEGEKTDKSKVKLTIAEDLSKTTFEIFKEDGKTLVSKKVTLKDKSSTEEKFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELKNALKLEHHHHHHB. garinii (strain DK29; OspA serotype 6) aa 18-274, lpplipidation signal sequence removed (MKATKLVLGAVILGSTLLAG,SEQ ID NO: 15), C-terminal His tag (LEHHHHHH), N-terminalCSSF for addition of lipids SEQ ID NO: 39CSSFKQNVSSLDEKNSVSVDLPGGMTVLVSKEKDKDGKYSLEATVDKLELKGTSDKNNGSGTLEGEKTDKSKVKSTIADDLSQTKFEIFKEDGKTLVSKKVTLKDKSSTEEKFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELKNALKLEHHHHHHChimeric OspA Serotype1/Serotype2, N-terminal lipidation,His-tagged, including the OspB lipidation signal sequence:MRLLIGFALALALIG (SEQ ID NO: 14) which is cleaved during processingSEQ ID NO: 40MRLLIGFALALALIGCAQKGAESIGSVSVDLPGEMKVLVSKEKDKNGKYDLIATVDKLELKGTSDKNNGSGVLEGVKTNKSKVKLTISDDLGQTTLEVFKEDGKTLVSKKVTSKDKSSTEEKFNEKGEVSEKIITMADGTRLEYTGIKSDGTGKAKYVLKNFTLEGKVANDKTTLEVKEGTVTLSMNISKSGEVSVELNDTDSSAATKKTAAWNSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALKLEHHHHHHChimeric OspA Serotype5/Serotype3, N-terminal lipidation, His-tagged, including the OspB lipidation signal sequence:MRLLIGFALALALIG (SEQ ID NO: 14) which is cleaved during processingSEQ ID NO: 41MRLLIGFALALALIGCAQKGAESIGSVSVDLPGGMKVLVSKEKDKNGKYSLMATVEKLELKGTSDKNNGSGTLEGEKTNKSKVKLTIAEDLSKTTFEIFKEDGKTLVSKKVTLKDKSSTEEKFNEKGEISEKTIVMANGTRLEYTDIKSDKTGKAKYVLKDFTLEGTLAADGKTTLKVTEGTVTLSMNISKSGEITVALDDTDSSGNKKSGTWDSDTSTLTISKNSQKTKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELKAALKLEHHHHHHChimeric OspA Serotype6/Serotype4, N-terminal lipidation, His-tagged, including the OspB lipidation signal sequence:MRLLIGFALALALIG (SEQ ID NO: 14) which is cleaved during processingSEQ ID NO: 42MRLLIGFALALALIGCAQKGAESIGSVSVDLPGGMTVLVSKEKDKNGKYSLEATVDKLELKGTSDKNNGSGTLEGEKTNKSKVKLTIADDLSQTKFEIFKEDAKTLVSKKVTLKDKSSTEEKFNEKGETSEKTIVMANGTRLEYTDIKSDGSGKAKYVLKDFTLEGTLAADGKTTLKVTEGTVVLSMNILKSGEITVALDDSDTTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELKNALKLEHHHHHH S1D1 SEQ ID NO: 43FNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEICNALK S2D1 SEQ ID NO: 44FNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALK S3D1 SEQ ID NO: 45FNDKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKTKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALK S4D1 SEQ ID NO: 46FNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALK S5D1 SEQ ID NO: 47FNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALK S6D1 SEQ ID NO: 48FNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALK S3HYBD1 (BVA) SEQ ID NO: 49FNEKGEVSEKILTRSNGTTLEYSQMTDAENATKAVETLKNGIKLPGNLVGGKTKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKTKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALK BVAD1 SEQ ID NO: 50FNEKGEVSEKILTRSNGTTLEYSQMTDAENATKAVETLKNGIKLPGNLVGGKTTLKITCGTVTLSKHIAKSGEVTVEINDTSSTPNTKKTGKWDARNSTLTIIVDSKNKTKLVFTKQDTITVQSYNPAGNKLEGTAVEIKTLQELCNALKS3hybD1(Bsp): hybrid OspA C-terminal fragment; amino acids126-175 from Borrelia spielmanii and amino acids 177-274from Borrelia garinii, strain PBr, with disulfide bond type1 and T in position 233 SEQ ID NO: 51FNEKGELSEKTLVRANGTKLEYTEIKSDGTGKAKEVLKDFTLEGTLANEKTKLTVT C GTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQK T KQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAEL C AALK MSPD1 SEQ ID NO: 52FNEKGELSEKTLVRANGTKLEYTEIKSDGTGKAKEVLKDFTLEGTLANEKATLTVKCGTVTLSKNIDKSGEVTVALNDTDSTAATKKTGAWDSKTSTLTITVNSKKTKDLVFTKQDTITVQKYDSAGTTLEGSAVEIKTLDELCNALKForward primer for the 16S-23S intergenic spacer SEQ ID NO: 53GTATGTTTAGTGAGGGGGGTG Reverse primer for the 16S-23S intergenic spacerSEQ ID NO: 54 GGATCATAGCTCAGGTGGTTAGForward nested primer for the 16S-23S intergenic spacer SEQ ID NO: 55AGGGGGGTGAAGTCGTAACAAGReverse nested primer for the 16S-23S intergenic spacer SEQ ID NO: 56GTCTGATAAACCTGAGGTCGGA Forward primer for the RecA gene of BorreliaSEQ ID NO: 57 CATGCTCTTGATCCTGTTTAReverse primer for the RecA gene of Borrelia SEQ ID NO: 58CCCATTTCTCCATCTATCTC25-mer peptide from the Invariable Region 6 (IR6) of VlsE SEQ ID NO: 59MKKDDQIAAAMVLRGMAKDGQFALK Mouse cathelin SEQ ID NO: 60RLAGLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPE KLK peptide SEQ ID NO: 61KLKLLLLLKLK N-terminal peptide for lipidation SEQ ID NO: 62 CKQN5′-(dIdC)₁₃-3′ SEQ ID NO: 63dIdC dIdC dIdC dIdC dIdC dIdC dIdC dIdC dIdC dIdC dIdC dIdC dIdC

The entire contents of all of the references (including literaturereferences, issued patents, published patent applications, andco-pending patent applications) cited throughout this application arehereby expressly incorporated by reference.

The invention claimed is:
 1. A nucleic acid encoding a polypeptidecomprising a first disulfide bond-stabilized C-terminal fragment of anouter surface protein A (OspA), wherein said first disulfidebond-stabilized C-terminal fragment is a hybrid C-terminal OspA fragmentconsisting of, from the N- to C-terminal direction, a fusion of a firstand a second OspA portion from two different Borrelia strains, whereinsaid polypeptide induces an immune response protective against aBorrelia infection, and wherein: i) said first OspA portion consists ofamino acids 125-176 or amino acids 126-175 of OspA from a Borreliastrain that is not the corresponding fragment of B. garinii, strain PBrOspA with SEQ ID NO: 8, wherein the numbering of amino acids isaccording to the numbering of corresponding amino acids of thefull-length OspA of B. burgdoferi s.s., strain B31 with SEQ ID NO: 5;and ii) said second OspA portion consists of amino acids 176-274 oramino acids 177-274 of OspA from B. garinii, strain PBr (SEQ ID NO: 8),wherein the second OspA portion differs from the corresponding wild-typesequence of SEQ ID NO: 8 at least by a) substitution of the wild-typeamino acid at position 183+/−3 of SEQ ID NO: 8 by a cysteine, and b)substitution of the wild-type amino acid at position 270+/−3 of SEQ IDNO: 8 by a cysteine, wherein a disulfide bond between the introducedcysteines is present.
 2. The nucleic acid of claim 1, wherein the hybridC-terminal OspA fragment consists of the amino acid sequence defined bySEQ ID NO:
 1. 3. The nucleic acid of claim 1, wherein said hybridC-terminal OspA fragment consists of the amino acid sequence defined bySEQ ID NO:
 51. 4. The nucleic acid of claim 1, wherein said second OspAportion comprises a substitution of a threonine residue at position 233of wild-type OspA of B. garinii, strain PBr as defined by SEQ ID NO: 8,with a proline residue.
 5. The nucleic acid of claim 1, wherein thepolypeptide further comprises a second disulfide bond-stabilizedC-terminal OspA fragment; wherein said second disulfide bond-stabilizedC-terminal OspA fragment consists of a C-terminal domain of an OspA fromB. burgdorferi s.s., B. afzelii, B. bavariensis, or B. garinii, whichdiffers from the corresponding wild-type OspA sequence at least by theintroduction of at least one disulfide bond, and wherein said seconddisulfide bond-stabilized C-terminal OspA fragment is not a hybridC-terminal OspA fragment.
 6. The nucleic acid of claim 5, wherein saidat least one disulfide bond is formed by the substitution of the aminoacid at position 182+/−3 of the wild-type sequence by a cysteine and bythe substitution of the amino acid at position 269+/−3 of the wild-typesequence by a cysteine; and wherein the numbering of said amino acids isaccording to the numbering of corresponding amino acids of the fulllength OspA of B. burgdorferi s.s., strain B31 (SEQ ID NO: 5).
 7. Thenucleic acid of claim 1, wherein the polypeptide comprises an E.coli-derived 1pp lipidation signal as defined by MKATKLVLGAVILGSTLLAG(SEQ ID NO: 15).
 8. The nucleic acid of claim 1, wherein the polypeptidecomprises a lipidation site peptide led by an N-terminal cysteineresidue as a site for lipidation, wherein said lipidation site peptideis defined by the amino acid sequence CSS.
 9. The nucleic acid of claim5, wherein the polypeptide comprises a linker between the hybridC-terminal OspA fragment and the second C-terminal OspA fragment,wherein said linker comprises the amino acid sequenceGTSDKNNGSGSKEKNKDGKYS (SEQ ID NO: 16).
 10. The nucleic acid of claim 1,further comprising a second hybrid C-terminal fragment of OspA.
 11. Thenucleic acid of claim 1, wherein said polypeptide comprises the aminoacid sequence of SEQ ID NO:
 27. 12. The nucleic acid of claim 1, whereinsaid polypeptide consists of the amino acid sequence of SEQ ID NO: 27.13. A nucleic acid encoding a polypeptide comprising the amino acidsequence of SEQ ID NO:
 27. 14. A vector comprising the nucleic acid ofclaim
 1. 15. A host cell comprising the vector of claim
 14. 16. The hostcell of claim 15, wherein said host cell is E. coli.
 17. The host cellof claim 16, wherein the E. coli is an E. coli BL21 cell.
 18. A methodfor producing a polypeptide, comprising the following steps: a) growingthe host cell of claim 15 under conditions allowing for expression ofsaid polypeptide, b) homogenizing said host cell to form a host cellhomogenate, and c) subjecting the host cell homogenate to purificationsteps.
 19. The method of claim 18, wherein said purification stepscomprise enriching the polypeptide in a lipid phase by phase separationand purifying over a gel filtration column.
 20. The method of claim 18,wherein said purification steps further comprise processing thepolypeptide over a buffer exchange column.
 21. The nucleic acid of claim1, wherein the nucleic acid comprises the nucleic acid sequence of SEQID NO:
 26. 22. The nucleic acid of claim 1, wherein the nucleic acidconsists of the nucleic acid sequence of SEQ ID NO:
 26. 23. A nucleicacid comprising the nucleic acid sequence of SEQ ID NO: 26.